Traceability of Cellular Cycle Ano

ABSTRACT

The present invention relates to the field of medicine and biology. It concerns a novel test for screening and for therapeutic follow-up in oncology. More particularly, it relates to diagnostic and/or therapeutic tests in oncology and on neurodegenerative diseases. It is a diagnostic test and a prognostic test for various cancers (breast cancer, bladder cancer, ovarian cancer, lung cancer, skin cancer, prostate cancer, colon cancer, liver cancer, glioblastoma, sarcoma, leukemia, etc.) and therapeutics solutions for specific neurodegenerative diseases. More particularly, the invention concerns the use of the LIV21 protein, LIV21 gene and of derivatives thereof as diagnostic and prognostic markers for cancers. The invention therefore concerns the detection of the LIV21 protein with a kit comprising LIV21-specific antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Divisional Application of U.S. patent application Ser. No.12/047,173 filed on Mar. 12, 2008 which is a continuation in partapplication of U.S. patent application Ser. No. 11/908,103 filed on Sep.7, 2007 which is a National Phase Entry of PCT Application Serial No.PCT/FR2006/000510 filed on Mar. 7, 2006 which claims priority fromFrench Patent Application Nos. 0502258 and 0502257 filed on Mar. 7,2005.

FIELD OF THE INVENTION

The present invention relates to the field of medicine and biology. Itconcerns a novel test for screening and for therapeutic follow-up inoncology. More particularly, it relates to diagnostic and/or therapeutictests in oncology and on neurodegenerative diseases.

DESCRIPTION OF THE PRIOR ART

Age-related neurodegenerative diseases and cancers both involve amodification of the physiological process of programmed cell death orapoptosis. Neuronal death is abnormally accelerated duringneurodegenerative diseases such as Alzheimer's disease, Huntington'sdisease, Parkinson's disease, etc. On the other hand, the cancerizationprocess corresponds to a blocking of apoptosis which results in anuncontrolled multiplication of cells. The link between these twoprocesses has currently become a major field of investigation inresearch on aging.

The control of the balance between cell division (mitosis),differentiation and programmed cell death (apoptosis) is fundamentalduring normal physiological processes, such as embryonic development,tissue regeneration and aging. An impairment of this balance can lead tomajor pathological situations such as the formation of tumors or certainneurodegenerative diseases.

Cancer is one of the principal causes of mortality throughout the world.Although, over the course of the last generation, the percentages ofdeaths related to cardiac and cardiovascular diseases and a large numberof other diseases has decreased, the number of deaths related to thevarious forms of cancer is on the increase.

Despite the rapid advance in our understanding of the various forms ofcancer, the low survival rates can generally be attributed to inadequatediagnosis and inadequate treatment. Most tumors can only be detectedwhen they reach a size of approximately 1 cm. Since there is arelatively short period of time from the continuous development of atumor to a stage which has become incompatible with survival, thisleaves little time for a therapeutic intervention. Early diagnosistherefore becomes the key to success for the treatment of cancer.

For a multitude of reasons, early diagnosis remains illusory for mostforms of cancer. For certain forms of cancer, disease-specific markersare not available or are only available at an advanced stage of thedisease, making diagnosis difficult. In certain other forms of cancer,the markers are available but are not always specific for the disease orthey may be associated with its benign form. In yet other cases, thetechniques exist but the prohibitive cost for applying them to thepopulation in general makes them unsuitable.

Skin cancer, for example, is the most widespread cancer in Canada. In1992 alone, 50 300 new cases of skin cancer were reported, compared with19 300 cases of lung cancer, 16 200 cases of colorectal cancer and 15700 cases of breast cancer. In other words, skin cancer is as common asthe three main types of cancer combined. Its incidence continues toincrease, with 64 200 new cases thereof in 1997, that is an increase of14 000 cases annually in 5 years. In particular, the incidence ofmalignant melanoma is increasing at a rate of 2% per year. Earlydiagnosis remains the key to an effective treatment. A malignant tumoris readily accessible and can be removed with minor surgery. In fact,recovery is 100% if skin cancer is detected early enough. The earlydiagnosis of skin cancer remains, however, difficult. The latter is notjust one disease but an entire range of conditions related to oneanother, which appear similar in many cases upon visual inspection. Adiagnosis on the basis of such an inspection is therefore subjective. Inorder to understand this subjectivity more fully, an abnormal skingrowth should be considered. This growth may be pigmented ornonpigmented. If it is nonpigmented and malignant, it is then probably abasocellular epithelioma or a spinocellular epithelioma. However, theclinical development of these two forms of cancer is very different. Abasocellular epithelioma spreads out laterally over the surface of theskin, without penetrating into the deeper skin layers. Thus, although itcan be disfiguring, a basocellular epithelioma rarely developsmetastases and is rarely fatal. However, a spinocellular epitheliomacauses metastases and is often fatal. It therefore becomes important tobe able to distinguish these two types of skin cancer. A definitivediagnosis of skin cancer requires a biopsy and histological analysis.However, the decision to send a biopsy for analysis (or even whether apatient should be referred to a dermatologist) becomes very subjective.There are several biopsies which are not taken although they should havebeen.

Colon cancer is the third most common cause of cancer-related mortalityin men and women in North America (16 200 cases per year). Earlydetection, leading to an early intervention, has demonstrated thattreatment success and survival rate can be improved. For example, the5-year survival rate is 92% for a patient whose disease was detected atan early stage, whereas the rate drops to approximately 60% in patientswith a localized cancer, and to approximately 6% in those withmetastases. However, only a third of colon cancers are detected at anearly stage. One of the reasons for this delay in diagnosis is theabsence of a sensitive, relatively inexpensive, non-invasive screeningtest.

Breast cancer is one of the most common cancers in women, with coloncancer. The mortality rate is the highest of all the cancers affectingwomen.

There are very few diagnostic markers capable of detecting breast cancerand they only have a predictive value of 20%. There are no markers,either, which can detect or determine the invasiveness or theaggressiveness of metastatic cancer cells.

Over the last few years, considerable progress has been made in theunderstanding of the means used by oncogenes and tumor suppressor genesfor regulating cell proliferation and apoptosis. One of the main targetsof these regulators is the family of E2F-type transcription factors inthe E2F and RB protein signaling pathway. These proteins play a centralrole in controlling cell division by coupling the regulation of thegenes required for progression of the cell cycle with extracellularsignals (mitogens, proliferation inhibitors). It behaves as an oncogeneby stimulating tumor cell proliferation.

Among the expressed genes are found:

-   -   overexpression of the E2F4 transcription factor and the c-myc        oncogene which induce apoptosis of post-mitotic cells by        accumulation of oxygenated reactants (Tanaka, 2002);    -   the p53 gene, which belongs to the tumor suppressor gene family,        blocks the cell cycle in the case of DNA lesion. It has now been        demonstrated that this gene is also involved in the progression        of apoptosis (Oren, 1994; Yonish-Rouach, 1996);    -   the cyclin D1, one of the proteins constituting the regulatory        subunits of the kinases of the cell cycle, essential to the        progression of the cell cycle. This protein is also expressed        during apoptosis in various cell types (Han et al, 1996; Pardo        et al, 1996).

It would be desirable to have novel diagnostic methods which woulddetect the presence of cancer with greater specificity and which wouldmake it possible to distinguish between aggressive cancer cells having atendency to metastasize and those which are more localized which have alower probability of metastasizing. A marker which can therefore revealcell proliferation would be of great use.

SUMMARY OF THE INVENTION

The present invention concerns a novel test for screening forreinduction of the cell cycle targeting oncology. It is a diagnostictest and a prognostic test for various cancers (breast cancer, bladdercancer, ovarian cancer, lung cancer, skin cancer, prostate cancer, coloncancer, liver cancer, glioblastoma, sarcoma, leukaemia, etc.). Moreparticularly, the invention concerns the use of the LIV21 protein and ofderivatives thereof as diagnostic and prognostic markers for cancers.The invention therefore concerns the detection of the LIV21 protein witha kit comprising LIV21-specific antibodies.

A first objective of the present invention is to demonstrate a methodfor the detection and prognosis of cancer and of its metastaticpotential. Preferably, the cancer is selected from breast cancer,bladder cancer, ovarian cancer, lung cancer, skin cancer, prostatecancer, colon cancer, liver cancer, a sarcoma, a leukaemia andglyoblastoma, without being limited thereto.

One aspect of the present invention consists of the use of LIV21 andisoforms of LIV21 as a prognostic indicator for cancer. In fact, whenLIV21 is located in the cytoplasm, the cancer cells in the tissues areaggressive. Conversely, when the LIV21 gene expression product ispreferentially located in the cell nucleus, this is a prognosticindicator that the cells of the tissue are differentiated and quiescentand therefore non-invasive. The effectiveness of a cancer treatment canalso be monitored by the traceability of this protein, and of itsderivatives and ratios with the associated proteins.

Moreover, detection of protein kinase C epsilon (PKCε) is alsoadvantageous since it has been determined that PKCε phosphorylates theLIV21 protein in order to maintain it in the cytoplasm. Thus, asignificant increase in PKCε is indicative of the presence of cancercells. Moreover, the LIV21/PKCε ratio increases in the cytoplasmicfraction of cancer cells.

In addition, the detection of the E2F1 and/or E2F4 proteins isadvantageous. In fact, the LIV21 protein forms a complex with E2F4 whichis capable of inhibiting the expression of the E2F1 gene in the nucleus,E2F1 gene expression being a sign of cell proliferation. Thus, adecrease in the association of LIV21 with the E2F4 protein is indicativeof the presence of cancer cells. Similarly, the presence of the E2F1protein in the nucleus is indicative of the presence of cancer cells.

Consequently, the present invention concerns a method for the detection(in vitro or ex vivo) of cancer cells in a biological tissue sample (forexample, breast, ovary, endometrium, bladder, melanoma, prostate,glioblastoma, etc.) from patients, this method comprising the detectionof the product of expression of the LIV21 gene in the nucleus and/or thecytoplasm of the cells in the biological tissue sample from saidpatient, localization of said product of expression of the LIV21 gene inthe cytoplasm being indicative of the presence of cancer cells, andlocalization of said product of expression of the LIV21 gene in thenucleus being indicative of the presence of noncancer cells. Preferably,localization of said product of expression of the LIV21 gene in thecytoplasm is indicative of the presence of invasive and/or metastaticcancer cells.

Optionally, the method according to the present invention also comprisesthe detection of the product of expression of at least one gene selectedfrom the group consisting of the protein kinase C epsilon (PKCε) gene,the E2F1 gene and the E2F4 gene. The method can in particular comprisethe detection of the product of expression of two of these genes or ofthe three genes. Moreover, at least one of the ratios LIV21/PKCε,LIV21/E2F4 and LIV21/E2F1 can be determined in the present method. Thisratio can be determined in the cytoplasm and/or in the nucleus.Preferably, these ratios are determined in the nucleus. Preferably,these ratios are compared with those obtained in a normal cell.

The same is true of the detection of HDAC1, which has been shown to beinvolved in PML/SUMO1/Rb/HDAC-1 complexes. More generally, the HDACfamily plays a key role in the regulation of gene expression. When theHDACs are overexpressed, they bring about tumor suppressor genesilencing, hence the advantage of using HDAC inhibitors in therapy,combined with other inhibitors which regulate the metabolic cascadeinvolving the protein complex which contains LIV21. The level ofexpression of each enzyme or polypeptide of the SUMO/Rb/HDAC complex or,for certain cell types, of the PML/SUMO/Rb/HDAC complex is an additionalindictor of the proliferative state of the cell. Thus, in a specificembodiment, the method according to the present invention also comprisesthe detection of the product of expression of at least one gene selectedfrom the group consisting of SUMO1, Rb, HDAC and PML.

The methods according to the present invention also consist in using thedetection of the LIV21 protein in combination with all the proliferationmarkers and transcription factors which play a role in the cancerizationand neurodegeneration process. The method therefore also comprises thedetection of the product of expression of at least five genes selectedfrom the group consisting of RBP2, E2F4, E2F1, E2F2, SUMO1, HDAC1,cycE/cdk2, cdk1, CREB1, p300, Rb, PML, p107 and p130 of the pocketprotein family. Thus, the invention lies in the fabrication and the useof diagnostic antibody arrays (FIG. 2) comprising LIV21-specificantibodies and antibodies for the various proteins of theLIV21-associated complex according to the phases of the cell cycle, thatis, without restriction thereto, antibodies specific for RBP2, E2F4,E2F2, E2F1, SUMO1, SUMO3, HDAC1, cycE/cdk2, cdk1, CREB1, p300, Rb, PML,p107 and p130 of the pocket protein family (FIG. 1). In addition, thediagnostic arrays according to the present invention can compriseantibodies specific for NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, erk andCAF1. Ki67 and CAF1 (Amoulzy; Institut Curie) are nuclear markers whichsignal the proliferative state of many cancers. The protein arrays willmake it possible to study protein expression, protein interactions andpost-translational modifications, more particularly phosphorylations andmethylations of certain proteins, which signal a characteristic state ofthe diseased cell. The state of expression and of silencing of certaingenes is different in diseased cells and in normal cells. Moreover, theprotein interactions and the metabolism of the diseased cell aredifferent from those of the normal cell.

The other aspect of the present invention is the use of the proteinsmentioned above as markers for the invasiveness and the metastaticaggressiveness of cancer cells of the prostate, colon, bladder,melanoma, ovary, endometrium and cervix, and cancers in neurobiology,etc.

In one embodiment, the expression product of the genes is detected atthe protein level. Preferably, the protein is detected using a specificantibody. For example, the protein can be detected by Western blottinganalysis. In a preferred embodiment, it is detected byimmunohistochemistry, immunocytochemistry or radiography, or byperoxidase labelling by microfludiic technique (sonic or light as sersor Raman effect).

In one specific embodiment of the method comprising the detection of theexpression product of the PKCε gene, a significant increase in PKCε isindicative of the presence of cancer cells. Moreover, the method canalso comprise the determination of the LIV21/PKCε ratio in the nucleusand/or the cytoplasm. This ratio can be compared with that observed in anormal cell. An increase in the LIV21/PKCε ratio in the cytoplasmicfraction is indicative of cancer cells.

In another specific embodiment of the method comprising the detection ofthe expression product of the E2F4 gene, the method comprises thedetection of the association of LIV21 with the E2F4 protein, a decreasein this association in the cell nucleus being indicative of the presenceof cancer cells. Moreover, the method can also comprise thedetermination of the LIV21/E2F4 ratio in the nucleus and/or thecytoplasm. This ratio can be compared with that observed in a normalcell. In an additional embodiment of the method comprising the detectionof the expression product of the E2F1 gene, the presence of the E2F1protein in the nucleus is indicative of the presence of cancer cells.Moreover, the method can also comprise the determination of theLIV21/E2F1 ratio in the nucleus and/or the cytoplasm. This ratio can becompared with that observed in a normal cell. The method according tothe present invention allows in particular the detection of metastasizedcancer, therapeutic monitoring and/or recurrences following treatment. Asecond aspect of the invention concerns the human LIV21 protein and alsothe fragments thereof. More particularly, the present invention concernsa purified or recombinant, isolated human LIV21 protein. It concerns inparticular an isolated polypeptide (issue to NT032977.8, hs1 33153)having an apparent molecular weight of approximately 50-51 kD by Westernblotting analysis and of approximately 60 kD when it is sumoylatedand/or a polypeptide having an isoelectric point of 5.6 in its 50-51 kDform and nine polypeptides having apparent molecular weight 110 kD, 64kD, 51 kD, 50 kD, 49 kD, 30 kD, 17 kD, 16 kD, 15 kD in hot conditions(at 100° C.) having an isoelectric point between 5.6 and 11 in its formsand a polypeptide characterized by one of the chromatograms of FIGS. 3-6and a polypeptide comprising a peptide sequence selected from SEQ ID Nos1-55, preferably from SEQ ID Nos 1-5, or a sequence having 70%, 80% or90% identity to said sequences, and one of the peptide sequencesobtained by MALDI (FIGS. 7 and 8) and NanoLC-ESI-MS (FIGS. 6.1, 6.2 &9). In a preferred embodiment, the polypeptide comprises the two peptidesequences SEQ ID Nos 1 and 2. In an even more preferred embodiment, thepolypeptide comprises a third peptide sequence SEQ ID No 3 and/or afourth peptide sequence SEQ ID No 4 or a sequence having (70%), 80% or90% identity to said sequences. Optionally, LIV21 also comprises asequence selected from the sequences SEQ ID Nos 5-55 or a sequencehaving 70%, 80% or 90% identity to said sequences. Preferably, the LIV21protein comprises a leucine zipper motif, a basic domain characteristicof DNA binding domains, a nuclearization sequence, which not onlymediates DNA binding but also acts as protein protein interaction domainfor E2F2, E2F4, E2F1 (by similarity with E2F proteins family (FIGS. 10.1and 11.1&2), item for retinoblastoma protein, Bcl2, CD53 antigen) andcomprises also a Hand Domain, Zinc finger Domain (PDZ domain) and helixloop helix domain like TCFL4. The similarity of the sequence withtransferase domain showed also by similarity with TRAM1L1 (withtransmembrane helix and NDST3 for the (antisens) sequences of C8T7clones (FIG. 11.3) which matched also on chromosome 4q26 permits tounderstand the complex interactions of LIV21. Digestion of the LIV21protein with trypsin gives more than 54 peptides corresponding to themonoisotopic peaks among all the peptides as specified, FIGS. 3-6; FIGS.7 and 9; FIGS. 8.1 and 8.2 (SDS PAGE gels) and sequences (sens andantisens) of LIV21 gene are ID Nos 119 to 148.

A third aspect of the invention concerns an antibody which bindsspecifically to a polypeptide according to the present invention. Moreparticularly, the antibody can bind specifically to a polypeptidecomprising a peptide sequence selected from SEQ ID Nos 1-55, preferablyfrom SEQ ID Nos 1-5, or a sequence having 70%, 80% or 90% identity tosaid sequences. The present invention concerns in particular ananti-LIV21 serum produced by immunizing an animal or a human with apolypeptide according to the present invention, in particular apolypeptide comprising a peptide sequence selected from SEQ ID Nos 1-55,preferably from SEQ ID Nos 1-5, or a sequence having 70%, 80% or 90%identity to said sequences.

A fourth aspect of the invention concerns a kit for the detection ofcancer cells in a biological sample from a patient, this kit comprisingone or more elements selected from the group consisting of an antibodywhich binds specifically to human LIV21 according to the presentinvention and an anti-LIV21 serum according to the present invention. Ina specific embodiment of the invention, the kit also comprises means fordetecting the product of expression of five genes selected from thegroup consisting of the protein kinase C epsilon (PKCε) gene, the E2F1gene, E2F2 gene and the E2F4 gene. Preferably, the detection means is anantibody specific for the protein concerned. In another preferredembodiment, the kit also comprises a means for detecting the product ofexpression of a gene selected from the group consisting of RBP2, SUMO1,SUMO3, HDAC1, PML, cycE/cdk2, cdk1, CREB1, p300, Rb, p107, p130, NFkB,erk, Bcl2, CD53, cdc2A, mdm2, p21, p53, p65, Ki67 and CAF1. In apreferred embodiment, the kit comprises an antibody array comprising anLIV21-specific antibody. In a preferred embodiment, the array alsocomprises an antibody specific for a protein selected from PKCε, E2F1and E2F4. In addition, it can comprise five antibodies specific for aprotein selected from RBP2, SUMO, HDAC1, cycE/cdk2, cdk1, CREB1, p300,Rb, PML, p107 and p130 of the pocket protein family. The array can alsocomprise an antibody specific for a protein selected from NFkB, cdc2A,mdm2, p21, p53, p65, Ki67 and CAF1.

The invention concerns the use of an antibody specific for human LIV21for the diagnosis of cancer, and/or of five or more antibodies specificfor a protein complex containing LIV21, for example antibodies specificfor RBP2, E2F4, E2F2, E2F1, SUMO1, SUMO3, BRCA1, HDAC1, cycE/cdk2, cdk1,CREB1, p300, Rb, PML, erk, Bcl2, CD53, p107 and p130 of the pocketprotein family. Preferably, the diagnosis is performed ex vivo onsamples from a patient.

DESCRIPTION OF THE FIGURES

FIG. 1: antibody array.

FIG. 2: scheme of nuclear protein interactions and consequences on thestudy of therapeutics.

FIG. 3: LIV21 protein profile by mass spectrometry (Maldi) M (H⁺) forthe one-dimensional gel band corresponding to the protein doubletmigrating at 50 kD. The peptides derived from the digestion aresolubilized in a solvent: acetonitrile/water (1/1) containing 0.1% ofTFA (trifluoroacetic acid). A saturated solution of thealpha-cyano-4-hydroxycinnamic acid matrix is prepared in the samesolvent. The same volume of the two solutions is taken and mixedtogether, and 1 microliter is deposited onto the Maldi plate foranalysis. The spectrum was determined on a Voyager with Waters software.The calibration with respect to the autolysis and trypsin digestionpeaks is not excellent and needs to be looked at again.

FIG. 4 is a zoomed-in profile of the chromatogram of the 49 kD bandwithout smoothing.

FIGS. 5 & 5A is the second chromatogram corresponding to theone-dimensional 12% acrylamide gel band migrating at 53 kD (band 2) andrevealed with coomassie blue and the LIV21 antibody. The spectrum wasdetermined on a Brucker apparatus.

FIG. 6A is the third chromatogram corresponding to the one-dimensionalacrylamide gel band migrating at 49 kD (band 3) and revealed withcoomassie blue and the LIV21 antibody. The spectrum was also determinedon a Brucker apparatus.

FIG. 6B is an example of a chromatogram.

FIG. 6C is another example of a chromatogram.

FIG. 7A-7BB is a table of the monoisotopic peaks with a value M H⁺. Themasses are given with three numbers after the decimal point by theproteomic platforms since they estimate that this is the acquisitionprecision limit of MALDI TOF machines.

FIG. 8A represents a 2D SDS PAGE gel separating the twelve polypeptidesbound by the LIV21 antibody and

FIG. 8B represents the sample at 100° C. few minutes before migration.

FIGS. 9A-9C describes examples of Nano LC-ESI MS characterized peptides,including the peptides of the isoforms of LIV21. The table describes aNanoLC-ESI MS experiment. NanoLC makes it possible to separate thepeptides derived from the trypsin hydrolysis of the protein. The elutedpeptide fragments are ionized by electrospray and the ions formed aredetected by mass spectrometry (Q-TOF analyzer). Each of these ionscharacterizes a peptide specific for the protein. Legend illustrated bythe first page (FIG. 9A) describing the ion: molecular mass 523.25daltons. Title: elution from 29.15 to 29.23 corresponds to the peptideeluted between 29.19 and 29.23 minutes. By ionization, this moleculegives the double-charge ion (charge=2+): MH2⁺ of molecular mass m/z:523.25 daltons, hence the mass of the molecule M: 1044.51. The peptidesequence of this ion is determined by MSMS. The corresponding MSMSspectrum is defined by the column of numbers between Begin ions and Endions, which corresponds to an amino acid sequence (each amino acidhaving a specific mass, except for leucine and isoleucine, which havethe same molecular mass). The first column with a 4-decimal number(daughter ion: 86.1373) corresponds to the mass of the “daughter” ions.The second column (I: 45) corresponds to the intensity of these“daughter” ions.

FIGS. 9A-9C describes the MSMS analyses giving a set of polypeptidesthat can be assigned to the LIV21 protein and its complex orcontaminants according to the various observers of the varioussubcontracting proteomics platforms. Sequences common with Gallusgallus, the histatin variant HIS3-HUMAN, the HSP60 chaperonin, argininedeiminase, nucleotidyltransferase, dehydrogenase.

FIGS. 10A & 10B: Similarity between examples of peptides sequences LIV21comparison to E2F2.

FIG. 11.1A to 11.3C: Similarity between LIV21 and other genes as E2F2and N deacetylase/N-sulfotransferase and TRAML1.

FIGS. 12 & 12A: cartography: localization of LIV21 gene

FIG. 13A: alignments between the histatin-3-2 variant (by Brucker MaldiT of analysis), PATF and Q7TCL4 (turnip mosaic virus) AAN08045.2.

FIG. 13B: describes alignments between Gallus gallus (gi 50732569) andPATF and common polypeptide sequence (20 amino acids) derived from LIV21by MALDI TOF analysis.

FIG. 14 is the morphology of MCF7 cells treated or not treated with TPAat 25 nM.

FIG. 15 is an analysis by FACS; representation, for each phase of thecell cycle, of the percentage of cells as a function of treatment time:FIG. 15A. S phase, FIG. 15B. G2/M phase, FIG. 15C. G0/G1 phase. Thescale along the x-axis is not proportional to the duration of treatment.

FIG. 16 is a Western blot comparing total extracts (ET) and nuclearextracts (EN) and showing the inhibition, with TPA, of the expression ofLIV21 phosphorylation. FIG. 16A. as a function of the time of treatmentwith TPA at 25 nM; FIG. 16B. compared with LIV21 in protein extracts, at12 h of treatment with TPA at 25 nM.

FIG. 17 is a study of the nuclear translocation of LIV21 byimmunocytochemistry with an anti-LIV21 primary antibody (in green) incultures treated or not treated with TPA at 25 nM. The nuclei arestained red with propidium iodide. The nuclei are predominantly stainedyellow at 12 H until 24 H since the anti-LIV21 primary antibody (ingreen) is nuclear, whereas it is predominantly cytoplasmic at 72 H (rednuclei and green cytoplasms).

FIG. 18 shows the expression, as a function of time of treatment withTPA at 25 nM, of PKCε and PKCζ proteins in total extracts. α-Tubulinexpression serves as a control for the amount of total proteins loadedin the wells.

FIG. 19 shows the compared expression of PKCε and of LIV21 byimmunocytochemistry on MCF-7 cell cultures treated or not treated withTPA at 25 nM for 12 h, carried out with anti-LIV21 and anti-PKCεantibodies in green, and propidium iodide staining the DNA red. TheLIV21 is translocated into the nucleus by specific inhibition of PKCε.The PKCε is weakly expressed at 12 h in the presence of TPA. In fact,red nuclei and little green staining in the cytoplasms are observed. Onthe other hand, the expression of LIV21 is strong in the nuclei, whichare stained yellow (merge) both with the anti-LIV21 antibody (green) andwith the nucleus-specific propidium iodide.

FIG. 20 shows the effect of the PKCε-inhibiting peptide on the LIV21expression profile by immunocytochemistry on cultures: control ortreated with 1 μM of peptide, 2 μM of peptide, or 25 nM of TPA. Thetreatments last 12 hours. The cells are labeled with anti-LIV21 (ingreen), and with propidium iodide (in red). It is observed that 2 μM ofpeptide (image referred to as 2 μM) have the same effectiveness as “25nM of TPA 12 H”: the nuclei (yellow) are predominantly labeled both withpropidium iodide and with the anti-LIV21 antibody (in green) on thesetwo images, whereas the control and the cells treated with only 1 μM ofPKC-inhibiting peptide show red staining of the nuclei, reflecting theabsence of nuclear translocation of LIV21 through its anti-LIV21antibody (in green).

FIG. 21 shows the effect of the PKCε-inhibiting peptide on the LIV21expression profile in cytoplasmic (C) and nuclear (N) cellularfractions, after treatment for 12 h with TPA (25 nM) or with peptide (2μM).

FIG. 22 shows, by immunoprecipitation (IP), the coexistence betweenPML/SUMO and LIV21: nuclear yellow fluorescence (merge) corresponding tothe colocalization of PML/SUMO and LIV21 is observed in the cell nuclei.

FIGS. 23 and 24 show, by immunocytochemistry, the coexistence betweenPML/SUMO and LIV21. The colabeling (by fluorescent immunolabeling) ofLIV21 (green) and of SUMO-1 (red) in the nuclei of the cells treated ornot treated with TPA for 24 h and 72 h show that, at 24 h, LIV21 istranslocated into the nucleus where SUMO and PML coexist (merge: yellownuclei), whereas, at 72 h, LIV21 (green) is cytoplasmic. The nuclearbodies containing the SUMO1 protein are called PML bodies.

FIG. 25 shows that, using an array of 60 biopsies, including 50 of skincancer and 9 of normal tissues and a control T; nuclear LIV21 expressionis demonstrated in the biopsies of normal tissues and cytoplasmic LIV21expression is demonstrated in the biopsies of metastatic cancers.

Image 43: poorly differentiated skin cancer T2N0M0, image 58: normaltissue derived from the same individual suffering from a poorlydifferentiated skin cancer (the nuclei of the cells are stained yellow),image 40: 10 cm metastatic carcinoma, and image 17: metastatic carcinomaof 3.5 cm. The cell nuclei are stained red.

FIG. 26 is, like FIG. 25, a second example of nuclear localization ofLIV21 in the control and normal tissue (No. 52) (the cell nuclei arestained yellow), of the individual No. 7 suffering from a squamous cellcarcinoma of the pharynx (moderately differentiated T4N0M0). In theimages No. 7 of cancerous tissue, the cell nuclei are stained red.

FIG. 27 is a sample of advanced bladder cancer on cystectomy (grade IIIurethral carcinoma infiltrating the chorion and the musculosa) versusnormal bladder tissue from the same patient with an internal control(PI): preimmune serum PI before the rabbit has been immunized againstLIV21, red labeling of the nuclei with propidium iodide.

FIG. 28 is a sample of breast cancer at confocal microscopy with alexaand propidium coloration.

FIG. 29: Describes as FIGS. 5 and 6 the MALDI TOF analyses giving a setof polypeptide (the histatin-3-2 variant) that can be assigned to LIV21and its complex and contaminants which are sometimes different accordingto the observers of the various subcontracting proteomics platforms. TheMascot search parameters are: trypsin enzyme, variable modifications:carbamethylation and oxidation of methionines, without molecular masslimit, without isoelectric point restriction. Type of mass:monoisotopic. Mass error (MS): according to the observer 50 ppm or 100ppm. Non-cleavage with trypsin: 1 The masses captured are M (H⁺)/realmasses. For chromatogram 1, the cysteins are blocked with iodoacetamide.The possibility of digestion with Promega bovine trypsin may beincomplete with cleavage oversight.

Sequences common with Gallus gallus (gi 50732569), mouse syntaxin, thehistatin-3-2 variant (P15516-00-01-00), ZN575-Human, G6 P translocase,the HSP60 chaperonin, arginine deiminase, ferredoxin-NADP(+) reductase,pseudomonas polyribonucleotidyl transferase, dehydrolipoamidedehydrogenase.

FIG. 30: alignments between Gallus gallus (gi 50732569) and PATF andcommon polypeptide sequence (18 amino acids) derived from LIV21 by MALDITOF analysis.

FIGS. 31A & 31B: example of a Maldi analysis interpretation diagram forhistatin 3-2, giving sequence No. 5, selecting the masses: 2383.2610(delta at 0.005); 2539.3290 (delta: −0.02); 2511.3740 (delta at 0.02).score: 78 and expect: 0.0046.

FIG. 32: PCR with the primers showing a band of 1400 bp.

FIG. 33: gel 2 with analysis of molecular masses.

FIG. 34: gel 3 at 55° and analysis of molecular masses.

FIG. 35: gel 4 at 45° and at 55° and analysis of molecular masses.

FIG. 36: screening ligation of 400 bp band, clones B1 to B10.

FIG. 37: screening ligation of 1400 bp band, clones C1 to C10.

FIG. 38: gel 5: ligation screening on the five new clones.

FIG. 39: gel 6: screening of the S55T and S55M recombinant clones andanalysis of molecular masses.

FIGS. 40A-40B: examples of nucleotide sequence comparison between thesequenced Liv21 clones.

FIGS. 41A-41B: examples of nucleotide sequence comparison between thesequenced Liv21 clones.

FIG. 41: correspondence with sequences of Liv21 peptides: transductionof clones C8 and S55M1.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the identification of antigens in cell lysatesby immunoprecipitation. The analysis of the physical interaction ofvarious proteins associated with E2F4 and E2F1 has been studied bycoimmunoprecipitation of protein complexes. This analysis has made itpossible to demonstrate a novel marker which has a diagnostic andprognostic use for cancer.

A marker for PATF proliferation associated with the E2F family had beendemonstrated (Crisanti) through the characterization of exons, withoutthe gene having ever been cloned in humans nor a corresponding proteinhaving ever been found in humans.

The discovery of this novel molecule LIV21 could have a diagnostic valuefor the following reasons. By carrying out a screening of thelocalization of LIV21 in about ten human tumors, the inventor has beenable to observe that, in all proliferating tumor cells, this protein iscytoplasmic instead of nuclear. It is not therefore in the correctcellular compartment to be active on the arrest of cell multiplication.

It has thus been possible to observe the presence of LIV21 in mammals.The panel of LIV21 expression as a function of cell state (mitoticcycles, cell in the resting state, differentiation) has been studied ontissues originating from various mammals. Protein analyses on thevarious tissue samples have confirmed that the expression of thistranscription factor appears to be associated with a progression towarda quiescent cell state (arrest of mitoses and entry intodifferentiation). LIV21 is present in actively proliferating tumor celllines and its expression is essentially cytoplasmic. The same resultsare obtained on human mammary adenocarcinomas.

Thus, the present invention relates to a novel test for screening foranomalies of the reinduction of the cell cycle. This diagnostic test isbased on the study of the mechanism of action of the novel gene,encoding a potential novel transcription factor called LIV21, whichdown-regulate proliferation. LIV21 is implicated in the arrest of cellproliferation. LIV21 is cytoplasmic when the cells proliferate, whereasit becomes nuclear when the cells become quiescent. The characterizationof this factor suggests a new pathway for down-regulating cellproliferation, by virtue of its association with one of the members ofthe EF family: E2F4. The latter is known to down-regulate the cell cycleby association with the P130 protein or pocket protein of the RB family.

The localization observed for LIV21 in tumor cells (cytoplasmiclocalization) and in physiological cells (nuclear localization)suggests, in any event, that its function is disturbed when celldevelopment becomes anarchical.

The characterization of this molecule and the study of the timing andthe topology of its expression also indicate that the expression and thelocalization of this ubiquitous transcription factor are regulated as afunction of cell state: greater expression and nuclear localization forcells which have exited mitotic cycles, weak expression and cytoplasmiclocalization for actively proliferating cells such as human tumor cells.

LIV21 appears to be a key molecule for stabilizing another transcriptionfactor (E2F4) in the cell nucleus and thus for inducing an arrest ofcell proliferation may be to inhibiting E2F2 which active entrance ofviral sequences in breast cells.

Furthermore, it has been shown that the localization of LIV21 in thecytoplasmic compartment is regulated by PKCε. In fact, when LIV21 isphosphorylated by PKCε, LIV21 is located in the cytoplasmic compartment.Conversely, when the phosphorylation of LIV21 by PKCε is inhibited,LIV21 is located in the nuclear compartment.

The LIV21 gene is a human gene on chromosome 1p31 near FABP3 (MDGI),SERINC2 and TINAGL (P3ESCL) between [031,783,000-031,111, 000] with theneighbours which revealed a structure function relation (means some ofgenes which enter in interaction with LIV21 and the complexe LIV21 areneighbours (FIG. 12). For example, E2F2, Bcl2, CD53, HDAC1 are in thesame part of chromosome 1p.

LIV21

The present invention therefore concerns the LIV21 gene (FIG. 40.1), theLIV21 protein and derivatives and fragments thereof (FIG. 40.3).

The LIV21 protein is a human protein of approximately 300 amino acids.However, depending on the alternative splicing that it undergoes, itexists as at least three forms of different sizes and nine forms in hotconditions (FIG. 8). Moreover, it can be phosphorylated or sumoylated.It has an apparent molecular weight of between 50 kD and 51 kD inWestern blotting analysis. This apparent molecular weight is 60 kD whenLIV21 is sumoylated and in hot temperature conditions for sample (withthe most concentration of sample), the profile change and we observedapparent molecular weight for peptides (revealed helix structures) in arange of 110 kD and 15 kD (FIG. 8.2). In its 51 kD form, which may bephosphorylated or nonphosphorylated, its isoelectric point is 5.6 andits intensity is 13632 but with hot temperature and nuclear samplesseparated to cytoplasmic samples there are spots at basic isoelectricpoint (FIG. 8). This protein has been characterized by mass spectrometry(Maldi) (Example 1; FIGS. 3-13). It gives more than 54 peptidesfollowing digestion with Promega trypsin (FIG. 7). The characteristicsof the LIV21 protein are also described in FIGS. 3-13. Several specificpeptides of LIV21 have been characterized, and in particular the LIV21apeptide, the LIV21b peptide (SEQ ID No 2), the LIV21c peptide (SEQ ID No3), the LIV21d peptide and the LIV21e peptide (SEQ ID No 50). Thelongest sequence with homology with PATF (Cortunix Japonicus Bird) isthe LIV21e peptide (SEQ ID No 50 & 51) and PL MII (FIG. 30).

Other specific peptides of LIV21 (Liv21a to f) are described below inthe patent in listing in ASCII form.

For the purposes of the invention, a preferred LIV21 protein comprisesat least one sequence chosen from SEQ ID Nos 1-55 or a sequence having70%, 80% or preferably 90% homology with said sequence.

The isolation of LIV21 gene by RT PCR analysis (before to choose theoligonucleotides primers for amplification) begin by using the onedimensional and two dimensional gel electrophoresis analysis (FIGS. 33and 34), the protein samples corresponding to the putative protein andto the elements of the complex were extracted from the gels and digestedwith trypsin (Promega) in order to be analysed by MALDI (FIGS. 3 to 5)and ESI MS/MS mass spectrometry. The results, when put up againstproteomic databanks, made it possible to reveal several peptidesequences of interest, including some given as an example (FIGS. 6 and7), some being found in humans with very significant scores (splicing ofhistatin, etc., FIGS. 30 and 31), these sequences were used as primers(once reverse-transcribed to cDNA) for screening a library formed frombreast cancer-specific MCF7 cells (FIGS. 32 to 39).

The cloning made it possible to bring to the fore about twenty clonesout of the 150 clones obtained, of which ten clones were sequenced andcharacterize the new gene LIV21 (FIG. 32). Based on these sequences,siRNAs were determined in order to allow regulation of silencing typewithin this metabolic complex of interest so as to develop therapeuticapplications (SEQ N° 122).

In post-mitotic cells, apoptosis could correspond to an aborted attemptat mitosis. It is in this context that the application of LIV21 has beendeveloped. The inventor has identified sequences of the LIV21 gene.Using the LIV21 antibody on affinity columns it has been able to extractpeptides of the LIV21 protein; it has also used a second approach bymeans of a coimmunoprecipitation kit (Pierce) in order to have largeramounts of proteins (Example 1). Based on peptide sequences of the LIV21protein, obtained by mass spectrometry (Example 2), primers which makeit possible to amplify a cDNA fragment were designed (Example 3). Afterculturing and amplification of MCF7 line cells, extraction andpurification of RNAs, RT PCRs and cloning in a shuttle vector werecarried out, and then screening of the resistant colonies and sequencingmade it possible to reveal sequences characterizing the LIV21 gene(Examples 4 and 5). More than twenty characteristic clones out of 150clones were studied. The cDNA of these clones was used to screen alibrary prepared from the total mRNA of MCF7 cells. The sequence of thisnew product is a new transcription factor, the nuclear translocation ofwhich is correlated with the establishment of cell quiescence.Furthermore, it forms heterodimers with other transcription factors andit binds to DNA.

The gene of this protein is characterized by five main sequences (cf.insert of clones extracted to MCF7 cells in sequences listing) andsequences representing an alternative splicing.

Using Northern blotting, the inventor then followed the expression ofthis new product during development, from the embryonic stage. It wasobserved that the amount of the LIV21 protein increases as developmentprogresses, i.e. as a quiescent cell state becomes established. Throughthe same strategy, the inventor showed that the LIV21/E2F4 complexinhibited the expression of E2F1. This complex could correspond to a newpoint of control in the arrest of cell proliferation.

The LIV21 protein comprises a leucine zipper motif, a basic domainscharacteristic of DNA binding domains, a nuclearization sequence, zincfinger domain and helix loop helix with repetitive motifs.

The present invention concerns a purified or recombinant, isolated humanpolypeptide having a sequence comprising the sequence SEQ ID No 1 and/orSEQ ID No 2. Preferably, the polypeptide comprises the sequences SEQ IDNos 1 and 2. In a preferred embodiment, the polypeptide comprises (inaddition) a sequence selected from SEQ ID Nos 3-55, preferably from SEQID Nos 3-5, or a sequence having 70%, 80% or 90% identity to saidsequences. In a specific embodiment, it comprises a sequence selectedfrom one of the peptide sequences obtained by MALDI (FIG. 7) andNanoLC-ESI-MS (FIG. 9). The invention also concerns the two peptidesLIV21a (SEQ ID No 1) and LIV21b (SEQ ID No 2). The invention alsoconcerns a peptide having a sequence selected from SEQ ID Nos 3-55,preferably from SEQ ID Nos 3-5, or a sequence having 70%, 80% or 90%identity to said sequences. It also concerns peptides comprising atleast 10 consecutive amino acids of human LIV21, preferably at least 20,30 or 50 consecutive amino acids of LIV21.

The invention also concerns LIV21 derivatives of interest which are, forexample, fusion proteins in which LIV21 is fused to labeled proteinssuch as GFP. Moreover, the LIV21 protein can be labeled by any meansknown to those skilled in the art.

The present invention also concerns an antibody which binds specificallyto a polypeptide according to the present invention, preferably humanLIV21, or a fragment or a derivative thereof. In a specific embodiment,the antibody binds specifically to an LIV21a or LIV21b peptide. In apreferred embodiment, the antibody binds specifically to a polypeptidecomprising a sequence selected from SEQ ID Nos 1-55, preferably from SEQID Nos 1-5, or a sequence having 70%, 80% or 90% identity to saidsequences.

The antibodies may be polyclonal or monoclonal. They may be antibodyfragments and derivatives having substantially the same antigenicspecificity, in particular antibody fragments (for example, Fab, Fab′2,CDRs), humanized antibodies, polyfunctional antibodies, single-chainantibodies (ScFv), etc. The antibodies of the invention can be producedusing conventional methods, including the immunization of an animal andthe recovery of its serum (polyclonal) or of spleen cells (so as toproduce hybridomas by fusion with appropriate cell lines).

Said antibodies can be obtained directly from human serum or from serumof animals immunized with the proteins or the peptides according to thepresent invention. Methods for producing polyclonal antibodies fromvaried animal species including rodents (mice, rats, etc.), primates,horses, pigs, sheep, rabbits, poultry, etc., are described, for example,in Vaitukaitis et al. (Vaitukaitis, Robbins et al. 1971). The antigen iscombined with an adjuvant (for example, Freund's adjuvant) andadministered to an animal, typically by subcutaneous injection. Repeatedinjections can be carried out. Blood samples (immune serum) arecollected and the immunoglobulins are separated.

The present invention concerns an anti-LIV21 serum produced byimmunizing an animal with a polypeptide according to the presentinvention. In a specific embodiment, the animal was immunized with theLIV21a and/or LIV21b peptide. In a preferred embodiment, the animal isimmunized with these two peptides. The present invention also concernsan anti-LIV21 serum produced by immunizing an animal or a human with apolypeptide according to the present invention, in particular apolypeptide comprising a peptide sequence selected from SEQ ID Nos 1-55,preferably from SEQ ID Nos 1-5, or a sequence having 70%, 80% or 90%identity to said sequences. For example, the peptides can be coupled toa carrier protein such as hemocyanin, and then injected into an animal,for example a rabbit, for immunization. Polyclonal antibodies wereobtained using these two peptides by having immunized two rabbits andhaving bled one rabbit so as to have a preimmune serum.

Methods for producing monoclonal antibodies from various animal speciescan be found, for example, in Harlow et al. (Harlow 1988) or in Kohleret al. (Kohler and Milstein 1975). These methods include theimmunization of an animal with an antigen, followed by the recovery ofthe spleen cells, which are subsequently fused with immortalized cells,such as myeloma cells. The resulting hybridomas produce monoclonalantibodies and can be selected by limiting dilution so as to isolate theindividual clones. The antibodies can also be produced by selection fromcombinatorial libraries of immunoglobulins, such as those disclosed, forexample, in Ward et al. (Ward, Gussow et al. 1989).

The invention also includes the use of the antibodies according to theinvention for the detection and/or the purification of the human LIV21protein. In particular, the LIV21-specific antibodies can be used forthe detection of these proteins in a biological sample. They thusconstitute a means of immunocytochemical or immunohistochemical analysisof LIV21 expression on tissue sections. Generally for such analyses, theantibodies used are labeled in order to be detectable. As analternative, the antibodies can be indirectly labeled.

In a preferred embodiment, the antibodies are labeled. The labelsinclude radiolabels, enzymes, fluorescent, luminescent or chemicallabels, magnetic particles, gold labeling, biotin/avidin labeling,peroxidase labeling, etc.

The invention also includes a method for detecting the LIV21 protein ina biological sample, comprising a step of suitable treatment of thecells by any appropriate means which makes it possible to render theintracellular medium accessible, a step of bringing said intracellularmedium thus obtained into contact with an antibody specific for thehuman LIV21 protein and a step of demonstrating the LIV21-antibodycomplex formed, by any appropriate means. In specific embodiments, thecytoplasmic and/or nuclear extracts are prepared, and these extracts arebrought into contact with the antibody specific for the human LIV21protein.

Diagnosis

The present invention teaches the development of the diagnostic testwhich also makes it possible to monitor the evolution of a cellproliferation. In particular, the present invention makes it possible tomonitor the evolution of a cell proliferation on fresh cells or tissues,on frozen cells or tissues and on tissues processed, inter alia, withparaffin. The applications may be the diagnosis of cancer and also themonitoring of the evolution of a cell proliferation. Preferably, thecancer is selected from breast cancer, bladder cancer, ovarian cancer,lung cancer, skin cancer, prostate cancer, colon cancer, liver cancer, asarcoma, a leukemia and glioblastoma, without being limited thereto.

Four of these properties can be used: its passage from the cytoplasmiccellular compartment to the nuclear compartment, the property ofassociating with the E2F4 transcription factor in order to form acomplex which inhibits the expression of the E2F1 factor, and theability of LIV21 to translocate in the nucleus through specificinhibition of PKCε, the sumoylation of LIV21 when the latter is nuclearand integrated into PML bodies and its interaction with HDAC.

The predominantly cytoplasmic state of this protein in cases of cancer,compared with its nuclear location in normal cells, is a geographicaland structural difference which makes it possible, without the need fora fluorescent label, to differentiate spectral profiles of thefunctional pattern of cancerous tissue versus normal tissue, and thus tomake the diagnosis.

These results show that the cytoplasmic localization of LIV21 is anindicator of the aggressiveness and of the metastatic potential of thecancer. The detection of the LIV21 expression indicates the presence ofcancer cells, more particularly of invasive, aggressive and/ormetastatic cancer cells. These results also show that the nuclearlocalization of LIV21 is an indicator of normal quiescent cells or ofwell-differentiated tissues.

The invention concerns, moreover, methods for the diagnosis or prognosisof cancer which implement the detection of the cytoplasmic localizationof a transcription factor located in the nucleus in normal cells.

The present invention concerns a method for the detection of cancercells in a biological sample from a patient, comprising the detection ofthe product of expression of the LIV21 gene in the nucleus and/or thecytoplasm of the cells in the biological sample from said patient,localization of said product of expression of the LIV21 gene in thecytoplasm being indicative of the presence of cancer cells andlocalization of said product of expression of the LIV21 gene in thenucleus being indicative of the presence of noncancer cells. Preferably,localization of said product of expression of the LIV21 gene in thecytoplasm is indicative of the presence of invasive and/or metastaticcancer cells. The method preferably comprises a prior step of suitabletreatment of the cells contained in the sample by any appropriate meanswhich makes it possible to render the intracellular medium accessible.The method optionally comprises a step of comparison with a biologicalsample which does not contain cancer cells.

Optionally, the method according to the invention also comprises thedetection of the product of expression of at least one gene selectedfrom the group consisting of the protein kinase C epsilon (PKCε) gene,the E2F1 gene and the E2F4 gene. The method can in particular comprisethe detection of the product of expression of two of these genes or ofthe three genes. Moreover, at least one of the ratios LIV21/PKCε,LIV21/E2F4 and LIV21/E2F1 can be determined in the present method. Thisratio can be determined in the cytoplasm and/or in the nucleus.Preferably, these ratios are determined in the nucleus. Preferably,these ratios are compared with those obtained in a normal cell.

The method can also comprise the detection of the product of expressionof at least five genes selected from the group consisting of RBP2, E2F4,E2F1, SUMO1, SUMO3, HDAC1, cycE/cdk2, cdk1, CREB1, p300, Rb, PML, p107and p130 of the pocket protein family. It can also comprise thedetection of the product of expression of at least five genes selectedfrom the group consisting of NFkB, cdc2A, mdm2, p21, p53, p65, Ki67,erk, CD53 and CAF1. The method can comprise the detection of aninteraction between some of these proteins and/or the detection of aposttranslational modification of one of these proteins.

The method may in particular comprise the detection of the product ofthe expression of two of these genes or of the three genes. Moreover, atleast one of the ratios LIV21/PKCε, LIV21/E2F4 and LIV21/E2F1 may bedetermined in the present method. This ratio can be determined in thecytoplasm and/or in the nucleus. Preferably, these ratios are determinedin the nucleus. Preferably, these ratios are compared with thoseobtained in a normal cell.

In one embodiment, the expression product of the genes is detected atthe mRNA level, it being possible for the mRNA to be detected by anymeans known to those skilled in the art.

Thus, the method according to the present invention also relates to thedetection of a polynucleotide encoding the human LIV21 protein or afragment thereof, for example LIV21a and/or LIV21b. The polynucleotideencoding LIV21 may be an mRNA, a cDNA or a genomic DNA. Thepolynucleotides may be isolated from cells of the biological sample.They may also be obtained by a polymerase chain reaction (PCR) carriedout on the total DNA of the cells or else by RT PCR carried out on thetotal RNA of the cells or polyA RNAs.

The mRNA may be detected by an RT PCR analysis. For this, the methoduses a pair of primers specific for the expression product to bedetected, in particular LIV21, PKCε, E2F1 or E2F4. The term “specificpair of primers” is intended to mean that at least one of the primers isspecific for the expression product to be detected, i.e. that this pairof primers makes it possible to specifically amplify a fragment of thedesired mRNA. Preferably, the RT PCR analysis is carried out on nuclearand/or cytoplasmic extracts of the cells contained in the sample fromthe patient. Optionally, the RT PCR analysis may be a quantitativeanalysis. A pair of primers specific for LIV21 can be prepared on thebasis of the teachings of the present application. For example, the pairof primers may comprise the primers described in the sequences listing.

The pairs of primers specific for PKCε, E2F1 and E2F4 are well known bythose skilled in the art (Caroll J S 2000; Mundle S D 2003; Stevaux O2002; Cheng T 2002; Opalka B 2002).

The mRNA may also be detected by Northern blotting analysis. For this,the method uses a probe specific for the expression product to bedetected, in particular LIV21, PKCε, E2F1 or E2F4. A probe specific forLIV21 can be prepared on the basis of the teachings of the presentapplication. An example of a specific probe comprises the sequence SEQID No 50. Preferably, the Northern blotting analysis is carried out onnuclear and/or cytoplasmic extracts of the cells contained in the samplefrom the patient. The nucleic probe is labelled. The oligonucleotidelabelling technique is well known to those skilled in the art. Thelabelling of the probes according to the invention can be carried outwith radioactive elements or with non radioactive molecules. Among theradioactive isotopes used, mention may be made of ³²P, ³³P or ³H. Thenon radioactive entities are selected from ligands such as biotin,avidin, streptavidin or digoxigenin, haptens, dyes and luminescentagents such as radioluminescent, chemoluminescent, bioluminescent,fluorescent or phosphorescent agents. The probes specific for PKCε, E2F1and E2F4 are well known to those skilled in the art.

In a preferred embodiment, the expression product of the genes isdetected at the protein level. Preferably, the protein is detected usinga specific antibody. Thus, the method comprises a step of bringing thecells of the biological sample into contact with an anti-human LIV21antibody. The antibodies may be monoclonal or polyclonal. The anti-LIV21antibody can, for example, be an anti-LIV21 serum.

When the product of expression of one of the genes PKCε, E2F1 and E2F4must be detected, the method can use antibodies specific for the PKCε,E2F1 and E2F4 proteins, respectively. Polyclonal and monoclonalantibodies directed against PKCε, E2F1 and E2F4 are commerciallyavailable. By way of example, mention may be made of, for PKCε, a rabbitpolyclonal antibody (Santa Cruz Technology, sc-214), for E2F1, a rabbitpolyclonal antibody (Santa Cruz Technology, sc-860), and for E2F4, arabbit polyclonal antibody (Santa Cruz Technology, sc-866). Preferably,the antibodies are labeled, directly or by means of a secondaryantibody. The antibody labeling techniques are well known to thoseskilled in the art.

In a specific embodiment, the protein can be detected by Westernblotting analysis. The Western blotting analysis can be carried out onnuclear and/or cytoplasmic extracts of the cells contained in the samplefrom the patient. Briefly, the proteins are migrated in a gel and thenblotted onto a membrane. This membrane is then incubated in the presenceof the antibodies and the binding of the antibodies is optionallyrevealed using labeled secondary antibodies.

In another embodiment, the protein is detected by immunohistochemistry,immunocytochemistry or immunoradiography. These techniques are wellknown to those skilled in the art. The immunocytochemical analysis canbe carried out on whole cells originating from the sample or which arederived therefrom, for example by cell culture. It can also be carriedout on isolated nuclei. The immunohistochemical analysis can be carriedout on mammary tissue sections.

By way of illustration, an immunocytochemical analysis can include thefollowing steps. However, it is understood that other preparatorymethods can be carried out. Cells originating from the biological sampleare cultured, preferably on slides (Lab Tek, Nunc, Germany), and thenwashed with buffer and fixed with paraformaldehyde (for example, 4%). Asaturation step is preferably carried out by incubating the cells withbuffer S (PBS-0.1% Triton X100-10% FCS). The cells are then incubatedwith a primary antibody and are then washed and incubated with afluorescent secondary antibody, if necessary. The nuclei can be labeledwith propidium iodide (Sigma). The slides are mounted in moviol forobservation by fluorescence microscopy. Moreover, isolated nucleisampled during a nuclear extraction can be fixed with paraformaldehyde(for example, 4%). The suspensions of nuclei are deposited between aslide and cover slip and the observation is carried out by fluorescencemicroscopy and by confocal microscopy. The primary antibodies are, forexample, rabbit antibodies and the secondary antibodies are labeledantibodies directed against rabbit IgGs.

The present invention also concerns the use of a protein array fordetecting the expression of one or more of these proteins, and/or aninteraction between two or more of these proteins, and/or theposttranslational modification of one or more of these proteins.

In a preferred embodiment, the detection of the product of expression ofone or more genes or of the interaction between several proteins iscarried out by means of a protein array. Thus, a polypeptide accordingto the present invention, in particular LIV21 or a fragment thereof, oran antibody specific thereto, or a fragment or a derivative thereofwhich conserves the binding specificity, can advantageously beimmobilized on a support, preferably a protein array. Such a proteinarray is included in the invention. This array can also contain at leastone polypeptide selected from the group consisting of protein kinase Cepsilon (PKCε), RBP2, E2F4, E2F1, SUMO, HDAC1, cycE/cdk2, cdk1, CREB1,p300, Rb, PML, p107 and p130 of the pocket protein family or at leastone antibody specific for one of these polypeptides, or a fragment or aderivative thereof which conserves the binding specificity. The arraycan also comprise other polypeptides well known to those skilled in theart to be advantageous for the detection and/or the prognosis of acancer, or antibodies specific for said polypeptides. These polypeptidescan, for example, be selected from the following list: NFkB, cdc2A,mdm2, p21, p53, p65, Ki67 and CAF1.

The protein arrays according to the present invention can be preparedaccording to the techniques well known to those skilled in the art. Inpractice, it is possible to synthesize the attached polypeptidesdirectly on the protein array, or it is possible to perform an ex situsynthesis followed by a step of attachment of the synthesizedpolypeptide to said array. Moreover, the polypeptides or antibodies tobe attached can be purified from a cell. The supports include smoothsupports (for example, metal, glass, plastic, silicon, and ceramicsurfaces) and also texturized and porous materials. Such supports alsoinclude, but are not limited to, gels, rubbers, polymers and otherflexible materials. The supports do not need to be flat. The proteins orantibodies of the array can be attached directly to the support or canbe attached by means of a spacer or a linker.

In a specific embodiment, an LIV21-specific antibody or a fragment orderivative thereof which conserves the binding specificity isimmobilized on the solid support. Thus, this array provides a practicalmeans for measuring the LIV21 expression product. Preferably, the arraycomprises at least one antibody specific for a polypeptide selected fromthe group consisting of PKCε, RBP2, E2F4, E2F1, SUMO, HDAC1, cycE/cdk2,cdk1, CREB1, p300, Rb, PML, p107 and p130 of the pocket protein family,preferably PKCε, E2F1 and E2F4. The array also comprises at least oneantibody specific for a polypeptide known to those skilled in the art tobe advantageous for the detection and/or the prognosis of a cancer, forexample NFkB, cdc2A, mdm2, p21, p53, p65, Ki67 and CAF1. The array cancomprise an antibody or a fragment or derivative thereof which has thesame specificity.

The protein arrays according to the invention are also extremely usefulfor experiments in proteomics, which studies the interactions betweenthe various proteins. In a simplified manner, peptides representative ofthe various proteins are attached to a support. Said support is thenbrought into contact with labeled proteins and, after an optionalrinsing step, interactions between said labeled proteins and thepeptides attached to the protein array are detected.

“Protein array” is intended to denote a support to which are attachedpolypeptides or antibodies, it being possible for each of them to bepinpointed by its geographical location. These arrays differ mainly interms of their size, the material of the support and, optionally, thenumber of polypeptides which are attached thereto.

The protein arrays can also be useful for the screening of testcompounds.

The present invention also relates to a method for the detection ofcancer cells in a biological sample from a patient, comprising thedetection of the product of expression of the LIV21 gene in the nucleusand/or the cytoplasm of the cells in a sample of cells in the biologicalsample from said patient, which method is characterized in that itcomprises at least: (a) bringing said biological sample into contactwith a protein array as defined above, and (b) revealing, by anyappropriate means, antigen-antibody complexes formed in (a), for exampleby EIA, ELISA or RIA or by immunofluorescence. Other detection methodsare described in detail in the following document: US2004152212.

Methods applicable for the synthesis of protein arrays are described,for example, in the following patents:

WO2004/063719, WO2005/016515, US2005019828, WO03018773, US2002187464,U.S. Pat. No. 5,143,854, U.S. Pat. No. 5,242,974, U.S. Pat. No.5,252,743, U.S. Pat. No. 5,324,633, U.S. Pat. No. 5,384,261,US2006035387, US2005100947, US2005233473, WO0198458, WO0172458,WO0004382, WO0004389, WO9015070, WO9210092, WO9310161, WO9512808 andWO9601836, the content of these patents being incorporated into thepresent application by way of reference. For example, these proteinarrays can be fabricated according to conventional methods described(Lubman David M, QIAO TIECHENG Alex, Mathew ABY J etc.) or novel toolsfor the automation of hybridization and of reading, US2004152212 and YuXinglong US 2005019828 and novel supports which attach polypeptides,Claus Peter Klages et al. (example FIG. 2).

The biological samples originate from a patient potentially sufferingfrom cancer or for whom it has been established that said patient issuffering from cancer. “Biological sample” is intended in particular tomean a sample of the biological fluid, living tissue, tissue fragment,mucosity, organ or organ fragment type, or any culture supernatantobtained by means of taking a sample. The method according to thepresent invention can comprise a step of taking a biological sample fromthe patient. The detection step can be carried out directly on a tissuesection of the sample, or on a culture of cells originating from thesample, or on total cell extracts, nuclear extracts and/or cytoplasmicextracts. The sample from the patient can come from a puncture, abiopsy, ground cellular material, a bronchial aspiration, a blood sampleor a urine sample.

In a specific embodiment of the method comprising the detection of theproduct of expression of the PKCε gene, a significant increase in PKCεis indicative of the presence of cancer cells. More specifically, theamount of PKCε in normal cells is compared with the amount of PKCε inthe cells of the sample, and the significant increase is determined bymeans of this comparison. The method according to the present inventioncan optionally comprise the measurement of the LIV21/PKCε content. ThisLIV21/PKCε ratio increases in the cytoplasmic fraction of cancer cellscompared with normal cells.

In another specific embodiment of the method comprising the detection ofthe product of expression of the E2F4 gene, the method comprises thedetection of the association of LIV21 with the E2F4 protein, and adecrease in this association is indicative of the presence of cancercells. The detection of the association of LIV21 with the E2F4 proteincan be carried out by concurrent detection of LIV21 and of E2F4 and/orby the concurrent measurement of HDAC1. The method according to thepresent invention can optionally comprise the measurement of theE2F4/LIV21 content. This E2F4/LIV21 ratio decreases in the nucleus ofcancer cells compared with normal cells.

In an additional embodiment of the method comprising the detection ofthe product of expression of the E2F1 gene, the presence of the E2F1protein in the nucleus is indicative of the presence of cancer cells.The method according to the present invention can optionally comprisethe measurement of the E2F1/LIV21 content. This E2F1/LIV21 ratioincreases in the nuclear fraction of cancer cells compared with normalcells.

The method according to the present invention allows in particular thedetection of metastasized cancer, therapeutic monitoring and/orrecurrences following treatment and makes it possible to determine thedegree of invasiveness of a cancer. The specificity of the detection canbe related to the crossing over of information obtained through theexistence and the topography of LIV21 by all imaging and spectroscopymeans and obtained by combination with other known cancerologicalindicators via protein arrays or microarrays. Thus, the detection basedon LIV21 can be combined with the detection of other cancer markers, inparticular breast cancer markers, known to those skilled in the art.

In fact, the present invention concerns a method for the therapeuticmonitoring of an anticancer treatment in a patient suffering fromcancer, comprising the administration of the anticancer treatment tosaid patient and the detection of cancer cells in a biological samplefrom the patient, according to the method of the present invention. Adecrease in cancer cells will be indicative of the effectiveness of thetreatment. The detection of cancer cells in a biological sample from thepatient, according to the method of the present invention, can becarried out once or several times over the course of the anticancertreatment or after the anticancer treatment. Preferably, the biologicalsample originates from the tissue affected by the cancer treated.

Moreover, the present invention also concerns a method for the detectionof recurrences subsequent to an anticancer treatment of a cancer in apatient, comprising the detection of cancer cells in a biological samplefrom the patient, according to the method of the present invention. Thedetection of cancer cells in a biological sample from the patient,according to the method of the present invention, can be carried outonce or several times after the anticancer treatment. The detection ofcancer cells is indicative of recurrences. Preferably, the biologicalsample originates from the tissue affected by the cancer treated.

The present invention also describes a kit for carrying out a methodaccording to the invention. More particularly, the invention concerns akit for the detection of cancer cells in a biological sample from apatient, comprising one or more elements selected from the groupconsisting of an antibody which binds specifically to human LIV21according to the present invention and an anti-LIV21 serum according tothe present invention, an oligonucleotide probe specific for the LIV21mRNA and a pair of primers specific for the LIV21 mRNA. In a preferredembodiment, the kit comprises antibodies which bind specifically tohuman LIV21. In another preferred embodiment, the kit comprises anoligonucleotide probe specific for the LIV21 mRNA. It may also comprisea probe specific for a “housekeeping” gene. The kit according to thepresent invention can comprise reagents for the detection of anLIV21-antibody complex produced during an immunoreaction. Optionally,the kit according to the present invention also comprises means fordetecting the product of expression of at least one gene selected fromthe group consisting of the protein kinase C epsilon (PKCε) gene, theE2F1 gene and the E2F4 gene. This detection means can be antibodiesspecific for the protein, oligonucleotide probes specific for the mRNAconcerned and/or a pair of primers specific for the mRNA.

The present invention also relates to a diagnostic compositioncomprising one or more elements selected from the group consisting of anantibody according to the present invention and a serum according to thepresent invention, an oligonucleotide probe specific for the LIV21 mRNAand a pair of primers specific for the LIV21 mRNA.

The present invention also concerns a diagnostic composition comprisingone or more elements selected from the group consisting of an antibodyaccording to the present invention and a serum according to the presentinvention.

Anticancer Therapy

In the context of an anticancer therapy, it is possible to envisionincreasing the amount of LIV21 present in the nucleus. For this, thenuclear localization of LIV21 could be promoted, for example bydecreasing the activity of PKCε in the cancer cells and by using HDACinhibitors.

In another specific embodiment of anticancer therapy, it is possible toenvision decreasing the activity of PKCε in the cancer cells. Thisdecrease in activity can be produced by decreasing the activity of thePKCε protein or by decreasing its expression. A decrease in the activityof the PKCε protein can be obtained by administering PKCε-proteininhibitors to the cancer cells. The PKCε-protein inhibitors are wellknown to those skilled in the art. A decrease in the expression of thePKCε protein can be obtained by using antisenses or siRNA specific forthe PKCε gene. Kits are commercially available. Moreover, the techniquesconcerning inhibition by means of antisense or siRNA are well known tothose skilled in the art (Arya R 2004, Lee W 2004, Sen A 2004, Platet N1998, Hughes 1987).

The present invention therefore concerns a pharmaceutical compositioncomprising a PKCε-protein inhibitor. It also concerns the use of apharmaceutical composition comprising a PKCε-protein inhibitor as amedicament, in particular for the preparation of a medicament for use intreating cancer. Finally, it concerns a method for treating cancer in apatient, comprising the administration to the cancer cells of aPKCε-protein inhibitor, the pKCε-protein inhibitor making it possible toreduce or abolish the cancerous phenotype of the treated cells. In afirst embodiment, the PKCε-protein inhibitor decreases the activity ofthe PKCε protein. In a second embodiment, the PKCε-protein inhibitordecreases the expression of the PKCε protein. Preferably, cancer isselected from breast cancer, bladder cancer, ovarian cancer, lungcancer, skin cancer, prostate cancer, colon cancer, liver cancer, asarcoma, a leukaemia and glioblastoma, without being limited thereto.

In the context of a therapy for a neurodegenerative disease, it ispossible to envision decreasing the amount of LIV21 present in thenucleus of the cells affected by the neurodegenerative disease. Thecells affected by the neurodegenerative disease are generally neurons,motor neurons, etc. In a preferred embodiment, the neurodegenerativedisease is chosen from Alzheimer's disease, Huntington's disease,Parkinson's disease and amyotrophic lateral sclerosis (ALS). For this,the nuclear localization of LIV21 could also be hindered, for example byincreasing the activity of PKCε in the cells affected by theneurodegenerative disease.

The inhibition or the blocking of LIV21 expression can be carried out byany means known to those skilled in the art. In particular, by way ofillustration, mention may be made of the antisense strategy, siRNA andribozymes. Thus, an antisense oligonucleotide or an expression vectorencoding this antisense oligonucleotide could be prepared and used toblock the translation of the mRNA encoding LIV21 n-vivo. Moreover, aribozyme can be prepared for cleaving and destroying, in vivo, the mRNAencoding LIV21. It is also possible to envisage a triple-helix strategyin which an oligonucleotide is designed so as to hybridize with the geneencoding LIV21 and to thus block the transcription of this gene.

Moreover, the nuclear localization of LIV21 could also be madeunfavourable, for example by increasing the activity of PKCε in thecells affected by the neurodegenerative disease. We observed a strongsimilarity of LIV21 with ADC7 neuronal thread protein (O60448 HUMAN)which is knew to be implicated in Alzheimer disease and find in thecerebrospinal fluid. So regulate an interaction between these twoproteins with therapeutic peptides or siRNA is interesting for thetreatment of Alzheimer disease.

In another specific method of therapy against a neurodegenerativedisease, it is possible to envisage increasing the activity of PKCε inthe cells affected by the neurodegenerative disease and permitsapoptosis. The cells affected by the neurodegenerative disease aregenerally neurons, motorneurons, etc. In a preferred embodiment, theneurodegenerative disease is chosen from Alzheimer's disease,hungtington's disease, parkinson's disease and amyotrophic lateralsclerosis (ALS). For this, the nuclear localization of Liv21 could alsobe hindered, for example by increasing the activity of PKCε in the cellsaffected by the neurodegenerative disease.

This increase in activity can be produced by increasing the activity ofthe PKCε protein or by increasing its expression. An increase in theactivity of the PKCε protein can be obtained by administeringPKCε-protein activators to the cells affected by the neurodegenerativedisease. The PKCε-protein activators are well known to those skilled inthe art (Toma O (2004), Activation of PKCs by DAG, AGPI: oleic acid,linoleic acid, arachidonic acid, etc. Activation and proteolysis of PKCsin gonadotropic cells: Communication 2004 by Macciano H, Junoy B, Mas JL, Drouva S V, UMR6544 Marseille). An increase in the expression of thePKCε protein can be obtained by using expression vectors encoding thePKCε protein and which make it possible to overexpress it in the cellsaffected by the neurodegenerative disease.

Thus, the present invention concerns a pharmaceutical compositioncomprising a PKCε-protein activator or an expression vector encoding thePKCε protein. It also concerns the use of a PKCε-protein activator or ofan expression vector encoding the PKCε protein, for the preparation of amedicament for use in the treatment of a neurodegenerative disease.

Screening Method

The invention concerns methods for the selection, identification,characterization or optimization of active compounds which decrease cellproliferation, based on the measurement of the nuclear versuscytoplasmic localization of LIV21, or of the binding of the LIV21protein to the E2F4 protein.

In a first embodiment, the selection, the identification, thecharacterization or the optimization of active compounds of therapeuticinterest comprises bringing a candidate compound into contact with acell and determining the nuclear versus cytoplasmic localization of theLIV21 expression product. An increase in the nuclear localization ofLIV21 indicates that the candidate compound is active in terms ofdecreasing or abolishing cell proliferation. A decrease in the nuclearlocalization of LIV21 indicates that the candidate compound is active interms of treating or preventing a neurodegenerative disease.

In a second embodiment, the selection, the identification, thecharacterization or the optimization of active compounds of therapeuticinterest comprises bringing a candidate compound into contact with acell and determining the level of expression of the gene encoding thePKCε protein. A decrease in the expression of PKCε indicates that thecandidate compound is active in terms of decreasing or abolishing cellproliferation. An increase in the expression of PKCε indicates that thecandidate compound is active in terms of treating or preventing aneurodegenerative disease.

In a third embodiment, the selection, the identification, thecharacterization or the optimization of active compounds of therapeuticinterest comprises bringing a candidate compound into contact with acell and determining the level of LIV21/E2F4 complex. An increase in thelevel of LIV21/E2F4 complex indicates that the candidate compound isactive in terms of decreasing or abolishing cell proliferation. Adecrease in the level of LIV21/E2F4 complex indicates that the candidatecompound is active in terms of treating or preventing aneurodegenerative disease.

In a fourth embodiment, the selection, the identification, thecharacterization or the optimization of active compounds of therapeuticinterest comprises bringing a candidate compound into contact with acell and determining the level of expression of the gene encoding theE2F1 protein. A decrease in the expression of E2F1 indicates that thecandidate compound is active in terms of decreasing or abolishing cellproliferation. An increase in the expression of E2F1 indicates that thecandidate compound is active in terms of treating or preventing aneurodegenerative disease.

The invention also relates to a method of screening for a compoundcapable of interacting in vitro, directly or indirectly, with LIV21,characterized in that: in a first step, the candidate compound and LIV21are brought into contact and, in a second step, the complex formedbetween said candidate compound and LIV21 is detected by any appropriatemeans.

The present invention also relates to a method of screening for acompound capable of modulating (activating or inhibiting) the activityof the LIV21 protein, characterized in that: in a first step, cells of abiological sample expressing the LIV21 protein are brought into contactwith a candidate compound, in a second step, the effect of saidcandidate compound on the activity of said LIV21 protein is measured byany appropriate means, and in a third step, candidate compounds capableof modulating said activity are selected. The activity of LIV21 can, forexample, be estimated by means of evaluating the ability of the cell todivide, by measuring the expression of the E2F1 gene or by thecytoplasmic and/or nuclear localization of LIV21.

The candidate compound can be a protein, a peptide, a nucleic acid (DNAor RNA), a lipid, or an organic or inorganic compound. In particular,the candidate compound could be an antibody, an antisense, a ribozyme oran siRNA.

Other advantages and characteristics of the invention will appear in theexamples and the figures which follow, and which are given in anonlimiting manner.

EXAMPLES Example 1

The inventor performed mass spectrometry (MALDI) for the LIV21 proteinbased on a one-dimensional acrylamide gel. The LIV21 protein wasdigested with trypsin. The peptides derived from the digestion aresolubilized in a solvent: acetonitrile/water (1/1) containing 0.1% ofTFA (trifluoroacetic acid). A saturated solution of thealpha-cyano-4-hydroxycinnamic matrix was prepared in the same solvent.The same volume of the two solutions was taken and mixed together and 1μl was deposited onto the MALDI plate for analysis. The massspectrometry showed that the LIV21 protein digested with trypsin reveals54 peptides (cf. FIGS. 3-4). The LIV21 protein was characterized by amolecular weight of 50 kD, revealed by Western blotting. Since the firstMALDI results were not probative, the inventor produced atwo-dimensional SDS PAGE gel (FIG. 8). More than ten proteins wererevealed by silver nitrate staining, but the very small amount ofmaterial did not make it possible to test samples derived from this gelby MALDI or MSMS.

When it changes cell compartment and when it is sumoylated, the LIV21protein has a molecular weight of approximately 60 kD. When it isphosphorylated in the cytoplasm, it exhibits two forms which differ by afew kilobases. A doublet is then observed.

The inventor performed a third MALDI analysis, which gave interestingresults, especially with regard to the 49 kD gel band, on Gallus gallusand a histatin variant (FIG. 13), the inventor then examined thesequence alignments, which made it possible to confirm homologiesbetween Gallus gallus, PATF, Q7TCL4 and the polypeptides of histatin andof Gallus gallus (FIGS. 29 to 31.2).

Only two peptides characterize in common PATF and LIV21:

-   -   The LIV21a peptide is located between a site of interaction with        the Rb/p107/p130 protein (ITCCE) and a site of sumoylation by        SUMO1. The sequence of this peptide is the following:        PeptideLIV21a peptide is SEQ ID No 1    -   The LIV21b peptide is located between a sumoylation site (PKPG)        and a phospholipase C site (YVKI) followed by (KKKRK) NLS. The        sequence of this peptide is SEQ ID No 2    -   The LIV21c peptide is SEQ ID No 3    -   The LIV21d peptide SEQ ID No 4 are specific of LIV21.

Other peptides are provided in the sequences SEQ ID Nos 5-55.

Example 2 Study of the Nuclear Translocation of LIV21 in MCF-7 Cells

The study of the subcellular distribution of LIV21 in different tumorlines of various origins showed an exclusively cytoplasmic localizationof this protein. The mechanism(s) by which LIV21 could be translocatedinto the nucleus in order to act on the cell cycle was (were) shown.

The presence of putative sites for phosphorylation by protein kinases C(PKCs), based on the possible homology that PATF is thought to have withthe LIV21 sequence, directed the inventor's study toward a possibleinvolvement of these proteins with respect to its nuclear translocation.The inventor therefore chose to study the MCF-7 line treated with TPA,which is known to modulate PKCs.

In parallel to this work, the inventor also studied the expression andthe localization of cell cycle proteins implicated in the signalingpathway in which LIV21 could act.

The MCF-7 Cell Line

The MCF-7 line is a nonclonal human line of breast adenocarcinoma cells.During their differentiation induced by exogenous factors, these cellsdevelop a hypertrophy, membrane protrusions and a tendency to dissociatefrom one another. They acquire a secretory phenotype which ischaracterized by the appearance of numerous granules and of secretorycanaliculi.

In vivo, these cells are relatively nonmetastatic and this lowinvasiveness is thought to be due to a low constitutive activity of theprotein kinases C (PKCs) and to a relatively low level of expression ofprotein kinase C alpha.

This line is used in many studies on proliferation, differentiation andapoptosis. These studies use appropriate drugs, such as TNF for theinduction of apoptosis, or TPA (12-O-tetradecanoyl phorbol-13-SUMOate)for the induction of differentiation and therefore for the study ofdeparture from the cell cycle.

The Effect of TPA on the MCF-7 Line

TPA is a known activator of PKCs. It activates the growth of normalbreast cells, does not modify the proliferation of the cells of benigntumors from this same tissue, but drastically inhibits the proliferationof the cells of human mammary tumor lines such as the MCF-7 line. Itreduces the cell growth of this line by positively controlling thec-erb-2 receptor and negatively controlling the retinoic acid receptorα, which are both expressed in particularly large amount in these cells.TPA greatly and rapidly inhibits the expression and the function ofestrogen receptors (ERs) and it induces the time- and dose-dependenttranslocation of protein kinases C (PKCs) from the cytosol to themembranes.

Furthermore, TPA increases the migratory capacity of MCF-7 cells invitro and a short period of treatment of these cells with TPA inducescellular expansion and microtubule organization characteristic of theirdifferentiation.

Expression of LIV21 in MCF-7 Cells

Firstly, the inventor verified the expression of LIV21 in these cells atthe protein level.

The inventor tackled the study of the expression of the LIV21 proteinthrough the Western blotting technique, with an anti-LIV21 antibody, inMCF-7 cells compared with mammary tissues. The anti-LIV21 antibodieswere obtained by the method described below. In this line, LIV21 isexpressed, both in the mammary tissues and in the MCF-7 cells, in theform of a doublet which migrates at an apparent molecular weight of 50kDa.

Production of Purified Anti-LIV21 Serum

The specific peptide sequences are the sequences No. 1 and No. 2.

These peptides were injected into rabbits (NZ W ESD 75 female, 2.3 kg atday 0), in agreement with standard immunization procedures, such as:

Day STEPS OF THE PROCEDURE Day 0 Collection of a control serum (20 ml)First intradermal injection (1 ml/rabbit) 1 tube for two rabbits with 1ml of antigen + 1 ml Freund's Complete Day 14 Second intramuscularinjection (1 ml per rabbit) 1 tube for two rabbits with 1 ml ofantigen + 1 ml Freund's Incomplete Day 28 Third intramuscular injection(idem D14) Day 39 The test serum is collected (5 ml) and conserved at 4°C. (serum D39) Day 49 Four subcutaneous injections (1 ml) 1 tube for tworabbits with 1 ml of ag + 1 ml Freund's Incomplete Day 60 Test serumcollected (25-30 ml) and storage at 4° (serum D60) Day 77 Fifthintradermal injection (1 ml/rabbit) idem D49 Day 88 Test serum collected(5 ml) and storage at 4° (serum D88) Day 99 Test serum collected (25 ml)and storage at 4° (serum D98) Day 102 Test serum collected (25 ml) andstorage at 4° (serum D102) Day 104 Test serum collected (25 ml) andstorage at 4° (serum D104) Day 106 Test serum collected (25 ml) andstorage at 4° (serum D106) Day 109 Total collected by total collectionof blood and storage at 4° C. (serum D109)

The reactivity of the serum obtained was tested with respect to bindingof the peptide sequences No. 1 and 2.

At D60, the serum shows a good reactivity with each sequence. The serumproduced did not bind to any member of the E2F family.

The Effect of TPA in MCF-7 Cells

It has been described in the literature that TPA induces the arrest ofproliferation and the differentiation of tumor cells of the MCF-7 line.In order to validate the culture conditions, the cell growth wasmonitored (by counting) beforehand over three days of culture in thepresence or absence of TPA at a concentration of 25 nM (FIG. 14).

The cell counts demonstrate a variation in the growth kinetics betweenthe nontreated cultures and the TPA-treated cultures. In fact, from thesecond day of culture onward, the number of cells is significantlydifferent between the two treatment conditions, TPA already inducing thearrest of cell proliferation. After 3 days of treatment, the controlcultures have twice as many cells as the treated cultures. TPA at theconcentration of 25 nM therefore clearly inhibits proliferation underour culture conditions.

In parallel, the inventor was able to observe that the TPA-treated cellsrapidly acquire characteristics of differentiated mammary gland cells(FIG. 14): hypertrophy, membrane protrusions and tendency to dissociatefrom one another. However, in the period of time over which the cellswere studied, the secretory phenotype (appearance of granules and ofsecretory canaliculi) was not observed.

FACS Analysis of the Effect of TPA

These preliminary studies of the effect of TPA showed in:

S phase: The FACS study shows that the number of cells in the S phasedecreases and reaches a limiting value between 12 h and 24 h of TPAtreatment (FIG. 15A). However, without any further addition of TPA, thenumber of cells in the S phase increases again so as to return to theinitial state at 72 h of treatment.G2/M phase: The number of cells in the G2/M phase increases from thebeginning of the treatment, with a maximum observed at 12 hours (FIG.15B).G0/G1 phase: The number of cells in the G0/G1 phase is at a minimum at 6hours and at a maximum at 24 hours (FIG. 15C).

A maximum of cells in the S phase are therefore observed in the earlyperiods of the kinetics (0 h to 6 h), a maximum of cells in the G2/Mphase is observed at 12 h and, finally, a maximum of cells in the G0/G1phase is observed at 24 h. Finally, without any further addition of TPA,the cells return to the S phase at 72 h.

In conclusion, these results therefore show that TPA acts rapidly on thearrest of cell proliferation and its effect on the reduction of thenumber of cells in the S phase is optimal at 12 h. 72 h after the singleaddition of TPA, the cells reinitiate the cell cycle.

The Effect of TPA on the Nuclear Localization of LIV21

The results obtained by flow cytometry led the inventor to study, inparallel, the expression of LIV21 in nuclear extracts prepared after 12h, 24 h, 48 h and 72 h of TPA treatment (FIG. 16A). During thesekinetics, maximum anti-LIV21 immunoreactivity was observed from 12 h,was maintained up to 48 h and returned to its initial intensity after 72h of treatment. These data are to be compared with those obtained byFACS. The immunoreactivity of LIV21 significantly increases at 12 h, atwhich time the number of cells in the S phase is minimal. It lasts untilthe reinitiation of the cell cycle observed at 72 h.

Furthermore, it can be noted that this immunoreactivity is detected inthe form of a single band at an apparent molecular weight of 50 kDa,whereas it was expressed predominantly in the form of a doublet in thetotal extracts. In order to determine the form of LIV21 to which thissingle band corresponds, it was compared on the same Western blot with atotal extract and a nuclear extract at 12 h of treatment (FIG. 16B). Theresults obtained show that the nuclear form of LIV21 corresponds to thelower band of the doublet. These data suggest that the LIV21 proteinmight be in a different phosphorylation state according to the cellcompartment.

The results of the immunocytochemical study carried out using ananti-LIV21 antibody show that the nuclear translocation of LIV21 is at amaximum at 12 h (FIG. 17) and the localization of LIV21 is predominantlycytoplasmic at 72 h, which is in agreement with the Western blottingobservations. However, it is interesting to note that the expression ofLIV21 already begins from 1 h of treatment in certain cells, since theyare not synchronous.

All these observations show that the nuclear translocation of LIV21 isconcurrent with the decrease in the number of cells in the S phase.

Example 3 Study of the Influence of PKCs on the Nuclear Translocation ofLIV21

Effect of TPA on PKCε Expression

Western blotting study: Given that the protein sequence of LIV21 hasputative PKC phosphorylation sites, including two specific for PKCε, theinventor tested the variation in the expression of this PKC as afunction of the duration of TPA treatment. It was observed that TPA actsvery rapidly on PKCε expression, which decreases from 30 min (FIG. 18).The expression of PKCzeta (PKCζ) is used as an internal control since itis not sensitive to TPA.

Immunocytochemistry: In parallel, immunofluorescence experiments ontreated or nontreated cultures made it possible to demonstrate thisdecrease in PKCε concurrent with the nuclear translocation of LIV21(FIG. 19). It can be observed that PKCε disappears from the cytoplasmwhen the cells are treated with TPA and that LIV21 is detected in thenucleus.

To conclude, it is interesting to note that PKCε is weakly expressed at12 h, at which time the fewest number of cells in the S phase and themaximum nuclear translocation of LIV21 are observed.

Example 4 Study of the Specific Role of PKCε on the NuclearTranslocation of LIV21 Using a Peptide which Inhibits PKCε Function andTranslocation

In order to determine the specific action of PKCε on the translocationof LIV21, the cultures were treated with a peptide which is a selectiveantagonist of the function and the translocation of this PKCε (EAVSLKPT(SEQ ID No 118)), and the results were compared with those obtained withTPA treatment. This peptide is recognized by the enzyme and binds as amodified substrate at the level of its catalytic site. It cannot bephosphorylated and acts as a specific inhibitor of the activity of PKCε.

The effect of the selective inhibition of the activity of PKCε on thenuclear translocation of LIV21 was studied by immunocytochemistry. Theseexperiments were carried out on nontreated cultures or cultures treatedfor 12 h with TPA at 25 nM or with the peptide at two differentconcentrations, 1 and 2 μM (FIG. 20). The peptide used at theconcentration of 2 μM has an effect identical to that of TPA on thenuclear translocation of LIV21.

These results were supported by cell fractionation experiments oncultures treated with the PKCε-inhibiting peptide at 2 μM, compared withTPA-treated cultures (FIG. 21). The same LIV21 expression profile wasobserved in the form of a doublet in the cytoplasm and of a single bandin the nuclear fraction.

The specific inhibition of PKCε induces nuclear translocation of LIV21,thereby suggesting that LIV21 could be the target for PKCε, which wouldmaintain it in the cytoplasm in a phosphorylated form.

Example 5 Western Blotting Analysis

This example describes the conditions used for a Western blottinganalysis of cancerous breast cells.

The protein extracts are heated at 80° C. for 5 minutes in a Laemmlibuffer (pH 7.4, 0.06 M Tris, 3% SDS, 10% glycerol, 1 mM PMSF,β-mercaptoethanol). The migration is carried out by SDS-PAGE (sodiumdodecyl sulfate-polyacrylamide gel electrophoresis). 10 to 20 μg ofproteins migrate in a 12% polyacrylamide gel for 1 h under denaturingconditions (migration buffer: 25 mM Tris base, 192 mM glycine, 1% SDS,pH 8.3). The proteins are then transferred onto a nitrocellulosemembrane (Schleicher & Schuell) for one hour by liquid transfer, in atransfer membrane (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3).The membranes, saturated in PBS-0.1% Tween-0.1% Triton X100-5% skimmedmilk for one hour, are brought into contact with the primary antibodydiluted in PBS-0.1% Tween-0.1% Triton X100-1% milk at ambienttemperature with gentle agitation for one hour to two hours. Afterwashing, the peroxidase-coupled secondary antibody is incubated with themembranes for 1 h. Revelation is carried out by means of achemiluminescence reaction using the ECL kit according to the supplier'sprotocol (Amersham).

The Primary Antibodies Used are:

The anti-LIV21 serum which was produced using two synthetic peptidesbased on the sequence of LIV21: peptide LIV21a (SEQ ID No 1) and peptideLIV21b (SEQ ID No 2). The peptides were coupled to hemocyanin beforebeing injected into rabbits for the immunization. The polyclonalantibody was obtained from these two peptides by having immunized tworabbits and having bled one rabbit so as to have a preimmune serum (inorder to be sure that this antibody did not already exist in thisrabbit).

The rabbit anti-CDK2 polyclonal antibody (Santa-Cruz technology sc-163)diluted to 1/200.The mouse anti-p21 monoclonal antibody (Dako, M7202) diluted to 1/150.The mouse anti-p27 monoclonal antibody (Santa-Cruz technology sc-1641)diluted to 1/100.The rabbit anti-PKCε polyclonal antibody (Santa-Cruz technology sc-214)diluted to 1/200.The rabbit anti-PKCδ polyclonal antibody (Santa-Cruz technology sc-216)diluted to 1/200.The rat anti-α tubulin polyclonal antibody (Serotec, MCAP77) diluted to1/500.

The Peroxidase-Coupled Secondary Antibodies Used (Caltag) are:

The goat anti-rabbit IgG(H+L) F(ab′)2 antibody diluted to 1/2000.The goat anti-mouse IgG(H+L) F(ab′)2 antibody diluted to 1/2000.

Example 6

It was demonstrated that the LIV21 protein is associated with PML bodiesand that, during sumoylation, LIV21 goes from a molecular weight of 50kd to 60 kd. Colocalization of LIV21 with SUMO in the PML-SUMO/LIV21complex was shown by immunoprecipitation (FIG. 22).

Antitumor role of PML bodies: At the proliferation stage, there arevisualized modifications in the PML bodies since these PML bodiesdissociate and degrade: (speckles), proteins then become available inthe nucleus for ensuring transcription, proliferation, immune reactionsand everything that is required for gene transcription. It has beenshown that PML associates with SUMO and with HDAC-1 (histonedeacetylase 1) and that its complex acts on the expression of E2F1 andPML thus acts on the arrest of proliferation by blocking E2F1. Thus, thePML/HDAC-1 complex down-regulates E2F1 expression. PML associated withRb (p130) binds to the deacetylated histones and blocks E2F1 by bindingto the chromatin (FIG. 2).

In acute promyelocytic leukemias, PML is truncated and becomes a fusionprotein with the retinoic acid receptor. This fusion protein(PMLRARalpha) is due to a 15/17 chromosomal translocation. A newtreatment for this disease by combining arsenic and retinoic acid inorder to induce cancer cells into apoptosis has been reported in theliterature. The PML protein is thought to regulate proliferation incancers and lymphomas. The inventor has shown, by immunoprecipitation,the association SUMO-PML in which LIV21 is located.

In the above examples, it was shown that LIV21 is phosphorylated by PKCεand that TPA is an inhibitor of PKCs. The TPA-treated MCF-7 lines showan inhibition of cancerous proliferation and a cell differentiation, andLIV21 is translocated into the nucleus. If a PKCε-specific inhibitorypeptide was used, it was the activity and not the expression of PKCεwhich was inhibited.

During this TPA treatment (25 nM), when E2F4, p130 and LIV21 werestudied (green fluorescence) in the nuclei labeled (DNA) with propidiumiodide (red fluorescence) (FIGS. 23 and 24), the following wereobserved:

after 12 h, intranuclear green fluorescence signals with the samepattern for E2F4, p130 and LIV21;after 48 h, when the proliferation begins, E2F4 has a comparablelocalization; but at 72 h, it disappears from the nucleus (to thebenefit of E2F1).

By observing, by double labeling, the colocalization of PML and of LIV21at 24 h of TPA treatment (cf. merge: yellow fluorescence), it wasobserved that they are colocalized in the nuclei. At 48 h, thecolocalization between LIV21 and SUMO is also observed (cf. FIG. 23).The hypothesis is that SUMO, which binds to LIV21, in fact targets LIV21into the PML bodies and that LIV21 is involved in the PML/SUMO/Rb/HDAC-1complexes. Two different approaches were carried out in order todemonstrate that LIV21 is physically associated with PML and SUMO in thenuclear bodies, by immunoprecipitation (FIG. 22) and by colocalizationby immunocytochemistry (FIGS. 23 and 24) (Rb, p130 and p107 are pocketproteins which have the same binding site). The Rb proteins repress cellgrowth (Fabbro, Regazzi R, Bioch Biophys Res Comm 1986 Feb. 2; 135 (1):65-73).

When TPA is added only once, its action is exhausted after 72 h,proliferation begins again and, at this time, the PML bodies dissociate,break up and become speckles, thus leaving the proteins to ensuretranscription, proliferation, etc. LIV21 is no longer located in thenucleus since it is then rephosphorylated by PKCε and thus remainslocated in the cytoplasm.

Example 7 Study of the Expression of LIV21 in Breast Cancer Biopsies andSkin Cancer Biopsies

In order to determine whether the observations obtained above areapplicable to human tissues, a large number of skin cancer biopsiesobtained from patients were studied by immunohistochemical reaction withLIV21-specific antibodies. The immunohistochemical determination ofLIV21 protein expression was carried out on 60 biopsies from patients (9patients having a biopsy of normal tissue versus a biopsy of canceroustissue), the other biopsies corresponding to cancers which were more orless advanced and, for some, metastatic (cf. Lame superbiochipsLaboratories, Seoul, Korea). Moreover, some paraffin slides frompatients suffering from bladder cancer and from breast cancer were alsostudied.

Immunocytochemical Analysis Protocol:

Deparaffinize the slides.Rehydrate the tissues.Saturate the nonspecific sites and permeabilize the membranes.Add the antibody in a humid chamber.Reveal the antibody.Deparaffinize the slides under a hood.Two successive baths of toluene (rectapur Prolabo) 2×30 min or 2×20 min;then dehydrate the tissues with rectapur alcohol at 100% for 15 min;then rectapur ethanol at 95% for 10 min; then rectapur 70% for a further10 minThaw the antibody at the same time.Rehydrate the tissues gently in PBS supplemented with 10% fetal calfserum and 0.1% Triton.Saturate the nonspecific sites (for example, with ovalbumin) andpermeabilize the membranes.Rehydrate for one hour.Deposit one ml of this “PBS” per section in order to cover the slidewithout it drying out at any moment (when it is a slide with cells andnot tissues, half an hour is sufficient).Place the pane and the stainless steel cover and water below so as tocreate a humid chamber.Add the antibody in the humid chamber.Dilute the rabbit serum to 1/200 in 4 ml of PBS triton, so as tocontinue to permeabilize the membranes, and FCS.Place 1 ml on each slide and keep away from the light and avoidevaporation.Leave overnight or for a minimum of three hours.Then rinse with 1× normal PBS pH 7, carry out two washes of 5 to 10 minso that no trace of the first antibody remains.While preparing the Alexa 488 green probe (in the cold at 4° and in thedark) diluted to 1/250, therefore 10 μliter in 2.5 ml of PBS, still with10% FCS and 0.1% Triton, rest the slides on the plate. Cover the sectionagain with 2.5 ml in order to maintain a humid chamber for one hour, andthen wash with 1×PBS, pH 7.Wash with propidium iodide at 0.5 microgram per microliter to be dilutedto 20 microgram per ml and then again to 1/50, but this time, diluted in1×PBS alone (50 microliters per 2.5 ml of PBS). Drain while taking themout of the PBS and then dispense 2.5 ml of propidium iodide over thefour slides for one minute, followed by two rinses with simple PBS.Mount the slides in Moviol before reading.

All the results are summarized in Table 1 below.

TABLE 1 Expression of the LIV21 protein determined byimmunohistochemistry in 50 skin cancer biopsies and 9 normal tissuebiopsies “MIXED” NUCLEAR LIV21 CYTOPLASMIC LIV21 WEAKLY LIV21 TUMOR TYPENEGATIVE POSITIVE POSITIVE NORMAL TISSUES 6/9 3/9 0 FROM THE SAMEPATIENTS WELL- 18/21  2/21  1/21 DIFFERENTIATED CARCINOMAS MODERATELY1/7 5/7 1/7 DIFFERENTIATED CARCINOMAS POORLY  2/11  3/11  6/11DIFFERENTIATED CARCINOMAS CARCINOMA 0 2/9 7/9 METASTASES

For Example:

FIG. 25: Results: image 43: poorly differentiated cancer; image 58:normal tissue derived from the same individual suffering from a cancer;image 40: metastatic carcinoma 10 cm and image 17: metastatic carcinomaof 3.5 cm.

FIG. 26 is, like FIG. 25, a second example of nuclear localization ofLIV21 in the control and normal tissue (No. 52) of individual No. 7suffering from a squamous cell carcinoma of the pharynx (moderatelydifferentiated T4N0M0).

FIG. 27 is a sample of advanced bladder cancer on cystectomy (grade IIIurothelial carcinoma infiltrating the chorion and the musculosa) versusnormal bladder tissue from the same patient with an internal control(PI): preimmune serum PI before the rabbit has been immunized againstLIV21, labeling of the nuclei with propidium iodide.

FIG. 28 is a sample of breast cancer which makes it possible todemonstrate the cytoplasmic labeling of the LIV21 antibody in the cancercells.

These results show that the cytoplasmic localization of LIV21 is anindicator of the aggressiveness and of the metastatic potential of thecancer. The detection of LIV21 expression indicates the presence ofinvasive, aggressive and metastatic cancer cells. These results alsoshow that the nuclear localization of LIV21 is an indicator of normalquiescent cells, that is of well-differentiated tissues.

Example 8 Physical Interaction of LIV21 with the Proteins of the E2FFamily

Communoprecipitation experiments carried out using anti-LIV21, anti-E2F1and anti-E2F4 antibodies made it possible to demonstrate that LIV21associates with E2F4.

The members of the E2F family are transcription factors whose role hasbeen widely described in the literature as being key molecules in thepositive or negative control of the cell cycle (Slansky J E and FarnhamP J 1996; Helin K 1998 and Yamasaki L, 1998), by virtue of theirassociation with the pRb protein (Wu C L, Zukerberg L R, Lees J A 1995)or pocket proteins. E2F1 positively controls the cell cycle bytransactivating the promoter of the genes responsible for cellproliferation (DNA polymerase alpha, thymidine kinase, DHFR, etc.),whereas E2F4 is described as one of the members of the EF family whichnegatively controls the cycle. Furthermore, a high expression of E2F1 inembryonic mammary tissues has been shown (Espanel X, Gillet G 1998),whereas it is no longer expressed in post-mitotic mammary tissues, tothe benefit of a large increase in E2F4 expression (Kastner A Brun G1998).

The identification of antigens has been carried out in cell lysates byimmunoprecipitation. The analysis of the physical interaction of variousproteins associated with E2F4 and E2F1 was demonstrated bycoimmunoprecipitation of protein complexes. The complex was studiedusing μ MACS PROTEIN with MICROBEADS (MILTENYIBIOTEC). When lysates of Saureus are added, the proteins A interact with the Fc portion of thespecific antibodies and the immunocomplexes become insoluble and aretherefore recovered by centrifugation. After breaking of the bonds(heating) between AG/AB and protein A-rich membranes, Western blottingwas carried out. These results suggest that the LIV21/E2F4 complexappears to play an important role in establishing cell quiescence.

Example 9 Functional Interaction of LIV21 with the Proteins of the E2FFamily

It was demonstrated that blocking the expression of the LIV21 proteinwas correlated with a decrease in the expression of E2F4 and with anincrease in the expression of E2F1. In parallel, the functional aspectof the increase in E2F1 was verified by studying the transcription oftwo of its target genes, DHFR and DNA polymerase α.

Example 10 Reverse Transcriptions

After MCF7 cells (ATCC passage 15) had been thawed and cultured up to200 million, they were trypsinized and frozen at 80° C. The RNA wasextracted from two pools of 50 million cells with the Nucleospin RNA Lkit (Macherey Nagel) ref. 740.962.20, resulting in a pool 1 of 318 μgand a second of 182 μg. The poly A+ RNA was extracted from 313 μg oftotal RNA of pool 1 using the oligo Tex mRNA Midi kit (Qiagen) ref.70042.

I Reverse Transcription:

The RNA was reverse transcribed with the Fermentas Revert Aid H minus MMuLV Reverse Transcriptase, ref. EP0451 batch 1124, with 3.64 μg oftotal RNA and 0.45 μg of mRNA, according to the supplier's conditions,with an oligo dT primer. Reactions were carried out at 2 differenttemperatures at 45° C. and 55° C. so as to eliminate the RNA structuresthat may hinder reverse transcription.

II PCR

The PCRs were carried out with the reverse transcriptions as templates,initially with the primers A1+oligo dT. Nested PCRs were subsequentlycarried out on these first PCRs, with the primers A1+Splicing, A1+GDBR1,or ATG+Splicing, ATG+GDBR1.

PCR amplification was then carried out with the primers specific for thegenes to be detected, using the cDNAs obtained after oligo dT RT.

Enzyme: Fermentas Taq DNA polymerase. Thermocycler: Bio Rad iCycler.

The quality of the cDNAs was tested by amplification of GAPDH, b actinand Histone H3.3 housekeeping genes.

TABLE 1  Primers Amplified fragment Reference 5′-3′ sequence sizeHistone N 5′-GTG GTa aag cac cca gga a-3′ 347 bp Histone I (reverse)5′-gct agc tgg atg tct ttt gc-3′ Hum GAPDH sense5′-TGA AGG TCG GAG TCA ACG G-3′ 983 bp Hum GAPDH antisense5′-CAT GTG GGC CAT GAG GTC-3′ Hum b-actin sense5′-GGA CTT CGA GCA AGA GATGG-3′ 234 bp Hum b-actin antisense5′-AGC ACT GTG TTG GCG TAC AG-3′ LIV21 (A1) 5′- -3′ LIV21 (A2) 5′- -3′odT 5′-TTTTTTTTTTTTTTTTTTT-3′ ATG galgal 5′- -3′ Splicing sense 5′- -3′Splicing reverse 5′- -3′ G reverse 5′- -3′

TABLE 2 PCR mix 25 mM Mg⁺⁺ 10 mM Forward primer Reverse primer 10X (1.5mM final dNTP 10 μM 10 μM Taq (μl) cDNA H₂O buffer concentration) (μl)(μl) (μl) 5 U/μl (μl) 18.3 2.5 1.5 0.5 0.5 0.5 0.2 1

PCR Cycles

Denaturation: 94° C. 2 minutesDenaturation: 94° C. 30 secondsAnnealing: 52-55° C. 1 minute 35 cyclesElongation: 72° C. 1 minute 30Final elongation: 72° C. 7 minutes

Conservation: 4° C.

III Controls

The PCR Products were Subsequently Controlled on Agarose Gels andAnalysed with the Biocapt 11.01 Software from Vilber Lourmat.

FIG. 32: Analysis molecular masses gel 1FIG. 33: Gel 2 with analysis of molecular massesFIG. 34: Gel 3 at 55° and analysis of molecular massesFIG. 35: Gel 4 at 45° and 55° and analysis of molecular massesFIG. 36: Screening ligation of 400 bp band, clones B1 to B10FIG. 37: PCR with the primers showing a band at 1400 bp, clones C1 toC10FIG. 38: Gel 5: ligation, screening on the five new clonesFIG. 49: Gel 6: screening of the S55T and S55M recombinant clones andanalysis of molecular masses.

Gel 2:

PCRs carried out using templates from PCRs performed with the primersA1+oligo dT on the RTs carried out at 45° C. on the total RNA and thepoly A+RNA (messenger RNA). The primers used for these PCRs are A1+G orA1+Splicing reverse.

On the RTs carried out using the total RNA, a band of 1178 1253 bp isamplified with the primers A1+G and A1+Splicing reverse. The poly A RNAwas used to carry out the RT and is weakly observed at the size (FIG.33) of 1400 bp for amplification with the primers A1+G, and of 415 bpwith the primers A1+Splicing reverse. In the A1+splicing PCR product,there are other bands, of 860 and 233 bp (FIGS. 38 & 39).

Gel 3:

PCRs carried out using templates from PCRs performed with the primersA1+oligo dT on the RTs carried out at 45° C. on the total RNA and thepoly A+RNA (messenger RNA). The primers used for these PCRs are A1+GDBR1or A1+Splicing reverse (FIG. 34).

For the PCRs carried out on RTs performed at 55° C., the same overallpattern of bands as that obtained on the RTs performed at 45° C. isfound.

No specific amplification is observed when the poly A RNA was used tocarry out the RT. On the RTs carried out on the total RNA, a major bandof 1554 1609 bp is found with the primers A1+GDBR1 and A1+Splicingreverse. A band at the theoretical size of 1455 bp is expected for anamplification with the primers A1+GDBR1 and a band with the theoreticalsize of 415 bp is expected with the primers A1+Splicing reverse. In the2 profiles, very clear bands of 1900-2100 bp and of 1000-1300 bp arefound, but with a weaker intensity than that of the band of 1500-1600bp.

In the A1+splicing reverse PCR product, there is another major band, of263 bp.

Gel 4:

Nested PCRs carried out using templates from PCRs performed with theprimers A1+GDBR1 or A1+Splicing reverse on the RTs carried out at 45° C.on the total RNA and the poly A+RNA (messenger RNA). The primers usedfor these PCRs are ATG+GDBR1 or ATG+Splicing reverse (FIG. 35).

The nested PCRs carried out with the primers ATG+GDBR1 give bands at1213 bp (RT 45° C.) and 1559+1315 bp (RT at 55° C.); the expectedtheoretical size is 1455 bp. The PCRs carried with the primersATG+Splice reverse give more varied band profiles.

These PCRs carried out on other PCRs performed with the primers A1+GDBR1or A1+Splicing reverse.

The presence of a band of 400 bp is noted in the profiles obtained frommessenger RNA (the band obtained from the reverse transcription carriedout at 55° C. is of greater intensity).

The profiles of the ATG+Splice reverse PCRs carried with total RNA atthe start give a band of 424 437 bp of very strong intensity. Bands of614 and 783 bp of very strong intensity are also found in the profile ofthe RT 45 and a greater number of bands, but of weaker intensity, isfound in the profile of the RT 55, bands at 1118, 936 and 749 bp. Theproducts of these various PCRs were cloned and sequenced.

Example 12

The PCR products of lanes 2, 4, 6, 7 and 8 were ligated with the plasmidpGEMT Easy, Promega, T7 vector and the recombinant clones were screened(FIG. 38).

Lane 2: G45T ligationsLane 4: S45T ligationsLane 6: G55T ligationsLane 7: S55M ligationsLane 8: S55T ligations

The recombinant clones obtained were screened (after extraction of theplasmid DNA) by restriction with the Eco RI enzyme, the sites of whichborder the site of insertion of the PCR products into the pGEMT Easyvector.

Screening of the Recombinant Clones:

The first experiments had been carried out using the ten clones B andthe ten clones C, FIGS. 36 and 37, and the results of the sequences ofclones B2 and C8 are given in the following example, and exhibit, bysequence comparison between clones, great homology with the clones ofthe second series of experiments.

Gel 5: Screening of the S45T and G45T Recombinant Clones

Analysis of Molecular Masses

The screening of the clones with Eco RI shows that, out of the 9 S45Tclones, 3 have inserts of 100 bp, 216 bp and 410 bp.

On the G45T clones, out of the 6 clones tested, 3 have inserts of 57, 71and 148 bp (FIG. 38).

FIG. 39: Screening of the S55T and S55M recombinant clones

Analysis of Molecular Masses

The screening with Eco RI shows that, out of the 13 clones screened, 7have inserts of sizes between 239 and 637 bp.

The clones G45T5 (148 bp), S45T9 (410 bp), S45T3 (100 bp), S55M1 (491bp), S55T6 (251 bp) and S55T9 (637 bp) were extracted so as to besequenced.

Primers Used for Determining the Sequences of the Various Clones:

ATG galgal 5′-atgtatattatata-3′ INV COMP ttagatatataataSplicing reverse 5′-aaatat-3′ Splicing reverse 5′-t-3′ inv compG reverse 5′-TG-3′ G reverse 5′-AT . . . -3′ inv comp

Results:

Clone B2, the band which is approximately 400 bp is the same fragment asS55M1, which itself is the same fragment as S45T9. These fragments areapproximately between 400 and 450 bp. This is not surprising since theywere obtained after a nested PCR carried out with the splicing reverseprimer and the A1 primer (for the B2 fragment) and the ATG galgal primerfor the S45T9 and S55M1 fragments. These fragments were obtained withRTs carried out at various temperatures. On the other hand, the S55T9fragment is nevertheless approximately 600 bp, and a part (300 bp)exhibits quite strong identity with the other fragments cloned (cloneFLJ)

The inventor approached the study of the expression of the LIV21 proteinby the Western blotting technique, with an anti LIV21 antibody, in MCF 7cells compared with breast tissues. The anti LIV21 antibodies wereobtained by the method described. In this line, LIV21 was expressed,both in breast tissues and in the MCF 7 cells, in the form of a doubletthat migrates at an apparent molecular weight of 50 kDa.

In conclusion, these results suggest that the LIV21/E2F4 complex acts asa complex which inhibits the expression of the E2F1 gene. This complexcould correspond to a new point of control in the arrest of cellproliferation.

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1-33. (canceled)
 34. A pharmaceutical composition comprising aPKCε-protein inhibitor and a carrier, wherein said PKCε-proteininhibitor is selected from a group of esthers of phorbol.
 35. Thepharmaceutical composition as claimed 34, wherein said PKCε-proteininhibitor is a peptide listed in SEQ ID NO:
 118. 36. The pharmaceuticalcomposition as claimed 34, wherein said PKCε-protein inhibitor includesat least one of (a) an antisense nucleotide sequence to the nucleotidesequence for a PKCε gene listed in SEQ ID NO:122 and (b) a siRNAnucleotide sequence specific for the PKCε gene as SEQ ID N°
 122. 37. Thepharmaceutical composition as claimed in claim 34, further comprising anHDAC inhibitor.
 38. The pharmaceutical composition as claimed in claim34, wherein said composition is formed as a medicament for treating atleast one type of cancer.
 39. The pharmaceutical composition as claimedin claim 38, wherein said at least one type of cancer comprises any ofbreast cancer, bladder cancer, ovarian cancer, lung cancer, skin cancer,prostate cancer, colon cancer, liver cancer, a sarcoma, a leukaemia andglioblastoma.
 40. A method for treating cancer comprising the activityof administering a therapeutically effective amount of a composition aPKCε-protein inhibitor and a carrier, wherein said PKCε-proteininhibitor is selected from a group of esthers of phorbol.
 41. The methodas claimed in claim 40, wherein the activity of administering includesadministering a composition with a therapeutically effective amount of aPKCε-protein inhibitor including a peptide listed in SEQ ID NO:
 118. 42.The method as claimed in claim 40, wherein the cancer treated by saidactivity of administering includes at least one of breast cancer,bladder cancer, ovarian cancer, lung cancer, skin cancer, prostatecancer, colon cancer, liver cancer, a sarcoma, a leukaemia andglioblastoma.
 43. The method as claimed in claim 40, further comprisingadministering a therapeutically effective amount of an HDAC inhibitor.